GASTROENTEROLOGY

Thiamine Transport by Basolateral Plasma Membrane Vesicles

M. JAROSE,

Department of Internal Medicine, Veterans Affairs Medical Center and University School of Medicine, Ann Arbor, Michigan

of Michigan

hiamine (vitamin B,) is a quaternary amine consisting of a pyrimidine nucleus linked to a thiazole ring. As thiamine pyrophosphate, it plays a critical role in carbohydrate metabolism as a coenzyme in the decarboxylation of a-keto acids and in the hexose monophosphate shunt as a cofactor for transketolase; this latter reaction occurs predominantly in the liver.’ Previous studies, using isolated rat hepatocytes, have suggested that the hepatic uptake of thiamine is a saturable, Na+- and energy-dependent a thiamine analogue process. ‘s3Using dimethialium, that does not undergo phosphorylation, thiamine

T

Rat Liver

RICHARD H. MOSELEY, PANKAJ G. VASHI, SUZANNE CHRIS J. DICKINSON, and PATRICIA A. PERMOAD

Hepatic thiamine transport is thought to be a saturable, Naf- and energy-dependent process. However, the transport of this organic cation has not been examined in experimental models that allow direct characterization of carrier-mediated processes. Recently, a sinusoidal organic cation/H+ antiport was identified, using AP-methylnicotinamide as a marker. To determine whether thiamine is a substrate for this antiport, the characteristics of thiamine uptake were examined in rat liver basolatera1 membrane vesicles. An inwardly directed Na+ gradient had no effect on thiamine uptake as compared with an identical K+ gradient. An outwardly directed H+ gradient stimulated thiamine uptake as compared with pH-equilibrated conditions, and Hfdependent uptake was not the result of an H+ diffusion potential. Identical pH gradients stimulated uptake under voltage-clamped conditions, consistent with electroneutral thiamine/H+ exchange. Unlabeled intravesicular thiamine trons-stimulated [3H]thiamine uptake. Choline and imipramine cis-inhibited thiamine/H+ exchange; a series of other organic cations and thiamine analogues had no effect. Carrier-mediated [3H]thiamine uptake showed two saturable systems. In conclusion, a thiamine/H+ antiport is present on the sinusoidal membrane, distinct from Na+/H+ and NMN+/H+ exchange.

1992;103:1056-1065

transport into isolated hepatocytes, dissociated from intracellular phosphorylation, also showed saturability and inhibition in the presence of Naf-free media, ouabain, and 2,4-nitrophenol.4 Nevertheless, the use of isolated hepatocytes as an experimental model allows only an indirect characterization of this and other hepatic transport processes. The recent development of methods for the reproducible isolation and separation of purified sinusoidal and canalicular plasma membrane vesicles from rat live?-’ has resulted in considerable progress in the identification, characterization, and localization of transport systems at the membrane level in liver, rivaling the amount of information derived from work using other polarized epithelial cells, such as proximal tubular cells and enterocytes.’ In this regard it should be noted that, in the intestine, early studies using isolated intestinal loops and everted sacs showed that thiamine transport was blocked by the absence of Na+ and by ouabain inhibition of Na+,K+-adenosine triphosphatase (ATPase) activity.gl’0 However, using intestinal brush-border membrane vesicles, thiamine transport was found to be no different in the presence or absence of an inwardly directed Na+ gradient.“*” We recently identified and characterized a sinusoidal organic cation/H+ exchange using the naturally occurring quaternary amine, N’-methylnicotinamide (NMN) as a marker-l3 Working on the premise that an outwardly directed H+ gradient represents a more physiological driving force than an inwardly directed Na+ gradient for the uptake of a quaternary amine such as thiamine, we examined whether thiamine was a substrate for this sinusoidal organic cation/H+ exchanger, and, if not, what the driving forces for hepatic thiamine uptake were, using basolateral rat liver plasma membrane vesicles as an experimental model. 0 1992 by the American Gastroenterological 0016-5085/92/$3.00

Association

September 1992

HEPATIC THIAMINE TRANSPORT

Materials and Methods Materials 13H]Thiamine (1.2-20 Ci/mmol) was obtained from Moravek Biochemicals (Brea, CA); [3H]N1-methylnicotinamide chloride (2.8 Ci/mmol) was obtained from ICN Biomedicals (Irvine, CA). All other chemicals and reagents were purchased from Sigma Chemical (St. Louis, MO). All water used in preparing media was deionized, and all solutions were filtered through 0.22 pm Millipore filters (Millipore, Bedford. MA) before use. Valinomycin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) were stored in absolute ethanol (Aaper Alcohol and Chemical, Shelbyville, KY). When used, ethanol was also added to controls and the total concentration of ethanol in membrane vesicle suspensions was identical and did not exceed 0.25% (vol/vol). Vecuronium, procainamide ethobromide. and tributylmethylammonium were a generous gift from Dr. Dirk K. F. Meijer’s laboratory. Preparation

Membrane

of Rat Liver Plasma Vesicles

The method for isolating rat liver basolateral membrane (blLPM) vesicles, as well as their biochemical and morphological characterization, has been described in deafter isolation, memtail elsewhere.7,‘4,15 Immediately branes were suspended in the desired incubation media (exact composition is designated in the figure and table legends) at a protein concentration of 5-10 mg/mL and stored at -70°C for up to 1 month without loss of transport activity. Protein concentration was measured by the method of Lowry et a1.l6 using bovine serum albumin as standard. Interference of various buffer solutions with the protein assay was accounted for by determining separate standard curves for each buffer system. Transport

1057

Palo Alto, CA), and counted in a Beckman LS 1801 liquid scintillation counter. Nonspecific binding of isotope to filter and membrane vesicles was determined in each experiment by addition, at 0-4”C, of incubation medium and stop solution to 20 pL of membrane suspension. This membrane-filter blank was subtracted from all uptake determinations. Unless otherwise indicated, all incubations were performed in triplicate, and all observations confirmed with three or more separate membrane preparations. All values are expressed as mean f SE. The data were compared by Student’s t test; differences were considered statistically significant when P < 0.05. Reverse phase-high performance liquid chromatography was performed to assess the contribution of [3H]thiamine metabolism as a result of incubation with membrane vesicles to the observed transport measurements. A column (HR 5/5 PepRPC, FPLC, Pharmacia; Piscataway, NJ) was equilibrated with 20 mmol/L sodium phosphate/5 mmol/L tetrabutyl ammonium phosphate (pH 7.2) and eluted with an acetonitrile gradient. Fractions of 1 mL were collected and counted. The chromatographic profile in the presence of blLPM vesicles was superimposable to that in the absence of membrane vesicles (data not shown). Recovery of applied radioactivity was go%-100% in three separate experiments.

Results Initially, the effect on an inwardly directed Na+ gradient on [3H]thiamine uptake in blLPM vesicles was examined. As shown in Figure 1, uptake of 10 ymol/L [3H]thiamine over time in the presence of an inwardly directed 100 mmol/L Na+ gradient was

Measurements

Frozen membrane vesicle suspensions were rapidly thawed by immersion in a 37°C water bath, diluted to the desired protein concentration (3-5 mg/mL), and vesiculated by aspiration 10 times through a 25-gauge needle. Transmembrane transport of [3H]thiamine and [3H]NMN was measured by a rapid Millipore filtration technique. Membrane suspensions were preincubated at 25°C for at least 5 minutes before transport studies. Uptake into 20 PL of membrane vesicle suspension was initiated at 25°C by addition of 80 pL of reaction medium containing radiolabeled substrate. The exact composition of the reaction media is given in the figure and table legends of the individual experiments. After incubation for the designated time intervals, transport was terminated by the addition of 3 mL ice-cold stop solution, consisting of 204 mmol/L sucrose, 150 mmol/L K gluconate, 10 mmol/L Hepes-Tris (pH 7.5), 5 mmol/L Mg-gluconate and 0.2 mmol/L Ca-gluconate. Membrane vesicle-associated ligand was separated from free ligand by immediate filtration (1 mL/sec) through a 0.45pm Millipore filter (type HAWP; Millipore Corp., Bedford, MA) presoaked in stop solution. The filter was washed twice with 3 mL of stop solution. dissolved in Redisolv HP (Beckman Instruments,

TIME , minutes 1

Figure 1. Thiamine uptake in the presence of inwardly directed monovalent cation gradients. Membrane vesicles were suspended in 250 mmol/L sucrose, 100 mmol/L TMA gluconate, 0.2 mmol/L Ca gluconate, and 50 mmol/L HEPES/Tris, pH 7.5. Uptake of 10 pmol/L [3H]thiamine over time at 25’C was measured in media containing sucrose 250 mmol/L, 0.2 mmol/L Ca gluconate, 50 mmol/L HEPES/Tris, pH 7.5, and either 100 mmol/L NaCl or KCI. Data expressed as mean ? SE of quadruplicate analysis of three different membrane vesicle preparations.

GASTROENTEROLOGY Vol. 103, No. 3

1058 MOSELEY ET AL.

not significantly different from uptake in the presence of an inwardly directed 100 mmol/L K+ gradient. Uptake in the absence of any cation gradient (TMA) [ i.e., uptake under tetramethylammonium equilibrated conditions (TMAi,_Out; 100 mmol/L)] did not significantly differ from uptake in the presence of an inwardly directed Na+ or K+ gradient (data not shown). Thus, in contrast to findings in isolated hepatocyte+ but similar to findings in intestinal brush-border membrane vesicles,1*,‘2 thiamine transport in blLPM vesicles does not exhibit Na+ dependence. Next, the effect of an outwardly directed H+ gradient on [3H]thiamine uptake in blLPM vesicles was examined. As shown in Figure 2, uptake of 1 pmol/L [3H]thiamine over time in the presence of an outwardly directed H+gradient (pHi, 5.9 / pH,,, 7.9) was significantly greater than uptake under pH-equilibrated conditions. The pH gradient-dependent uptake of [3H]thiamine exhibited a transient “overshoot” (u pt a k e at 15 seconds was significantly greater than equilibrium values, P < 0.001) consistent with uptake against a concentration gradient. Intravesicular volume, as reflected by equilibrium values, was similar in the presence and absence of a

Figure 2. Thiamine uptake in presence and absence of an outwardly directed H+ gradient in blLPM vesicles. Membrane vesicles were suspended in either pH 5.9 media (containing sucrose 62 mmol/L, K+ gluconate, 100 mmol/L, MES 91 mmol/L, Tris 29 mmol/L, HEPES 14 mmol/L, and Ca gluconate 0.2 mmol/L) or pH 7.9 media (containing sucrose 70 mmol/L, K+ gluconate 100 mmol/L, Tris 70 mmol/L, HEPES 76 mmol/L, and Ca gluconate 0.2 mmol/L). Uptake of 1 pmol/L [3H]thiamine over time was measured in pH 7.9 media at 25°C. Data expressed as mean + SE of triplicate analysis of three different membrane vesicle preparations. **P < 0.005. Inset: uptake of 1 pmol/L [3H]thiamine over time in the presence of an outwardly directed H+ gradient; RZ = 0.96.

Table 1. Effect of Intravesicular in blLPM Vesicles Thiamine Time

PH 5.9i,/5.9,,,

5 set 10 set

0.34 * 0.09 0.63 * 0.13

15 set 30sec 60sec 2 min 5 min 60 min

0.56f 0.14 0.78f 0.07 0.65k 0.13 0.63k 0.08 1.02* 0.13 1.13+-0.09

pH on Thiamine Uptake uptake (pmol/mg prot) PH 5.9,,/7.9,,, 2.11 t 0.14O

1.97f o.15a 2.57zk0.22" 2.03f 0.16' 1.74* o.17a 1.69+ 0.13' 1.51f 0.13 1.47AZ0.10

NOTE. Membrane vesicles were suspended in pH 5.9 media (containing sucrose 82 mmol/L, MES 91 mmol/L, Tris 29 mmol/L, HEPES 14 mmol/L, K gluconate 100 mmol/L, and Ca gluconate 0.2 mmol/L). Uptake of 1 pmol/L [3H]thiamine over time was measured at 25°C in either pH 5.9 media or pH 7.9 media (containing sucrose 70 mmol/L, Tris 70 mmol/L, HEPES 76 mmol/L, K gluconate 100 mmol/L, and Ca gluconate 0.2mmol/L). Data expressed as mean ? SE of triplicate analysis of three different membrane vesicle preparations. “P < 0.001.

pH gradient. As shown in the inset, the time course of pH gradient-dependent [3H]thiamine uptake was linear for 6 seconds (R’ = 0.96, y = -0.01 + 0.19x). Therefore, in subsequent experiments, a 5-second incubation was used to determine initial uptake rates. As an additional control to exclude an effect of pH per se on [3H]thiamine uptake, uptake of 1 pmol/ L [3H]thiamine over time was examined in separate experiments under pH, 5.9/pH,,, 5.9 conditions. As shown in Table 1, thiamine uptake in the presence of an outwardly directed H+ gradient (pH, 5.9/pH,,, 7.9) was again significantly greater than uptake under these pH-equilibrated conditions. To determine whether [3H]thiamine is transported into the intravesicular space rather than bound to the membrane, [3H]thiamine binding to blLPM vesicles was assessed by determining the effect of medium osmolarity on [3H]thiamine uptake at equilibrium (Figure 3). As the osmolarity increased, pH gradient-dependent thiamine uptake diminished proportionately, as predicted if [3H]thiamine is transported into a closed intravesicular space. Extrapolation of the values for thiamine uptake to infinite osmolarity (where the intravesicular space is 0) shows a binding component of 0.24 pmol/mg prot, corresponding to -16% of equilibrium uptake under experimental conditions. The effect of thiamine on pH gradient-dependent uptake of [3H]NMN, a substrate for the recently described basolateral organic cation/proton exchangerI was next determined. As shown in Table 2, increasing concentrations of thiamine had no effect on the initial rates of pH gradient-dependent

September

I

0

HEPATIC

1992

I

1

1

2

11 OSMOLAIWI’Y

Figure 3. Effect of medium osmolarity on pH gradient-dependent thiamine uptake in blLPM vesicles. Membrane vesicles were suspended in pH 5.9 media (containing sucrose 82 mmol/L, K+ gluconate 100 mmol/L, MES 91 mmol/L, Tris 29 mmol/L, HEPES 14 mmol/L, and Ca gluconate 0.2 mmol/L). Sixty-minute uptake of 1 pmol/L [3H]thiamine at 25°C was measured in pH 7.9 media, containing K+ gluconate 100 mmol/L, Tris 70 mmol/L, HEPES 76 mmol/L, and Ca gluconate 0.2 mmol/L, and varying concentrations of sucrose to alter extravesicular osmolarity. Data expressed as mean + SE of triplicate analysis of three different membrane vesicle preparations. Regression line (R’ = 0.84; y = 0.24 + 0.54X) calculated by least squares analysis. Dotted line represents extrapolation to theoretical 0 intravesicular volume.

NMN uptake. Equilibrium uptake values were likewise unaffected by the presence of thiamine. These findings suggest that pH gradient-dependent thiamine uptake is mediated by a transport system separate from NMN+/H+ exchange. Enhanced uptake of thiamine in the presence of an outwardly directed H+ gradient may have been the result of an intravesicular negative H+ diffusion potential. In other words, passive H+ movement outward in response to the imposed pH gradient may have resulted in a transient negative intravesicular space that would then drive the uptake of positively charged species. This possibility was examined by determining the effect of the proton ionophore, FCCP, on pH gradient-dependent thiamine uptake. The addition of FCCP should enhance passive H+ movement outward by creating a proton-selective leak in the membrane vesicle. As shown in Figure 4, in the presence of FCCP, pH gradient-dependent thi-

THIAMINE

TRANSPORT

1059

amine uptake was not further stimulated as would be expected if uptake was the result of an H+ diffusion potential. In contrast, inhibition of the initial rates of thiamine uptake was observed in the presence of FCCP, suggesting that the protonophore causes a more rapid dissipation of the pH gradient driving thiamine uptake. Previous studies showing stimulation of electrogenic Na+-alanine cotransport in blLPM vesicles by FCCP confirm that an intravesicular-negative H+ diffusion potential is achieved under these conditions.13 The effect of an outwardly directed H+ gradient on thiamine uptake in blLPM vesicles was next examined under voltage-clamped conditions (Kfin = Kfout , in the presence of the K+ ionophore, valinomycin, 5 pg/mg protein). In this manner pH gradient-dependent thiamine uptake can be studied in the absence of changes in membrane potential. As shown in Figure 5, thiamine uptake over time in the presence of an outwardly directed H+ gradient was unchanged by the addition of valinomycin. Under voltageclamped conditions, pH gradient-dependent thiamine uptake still showed a transient “overshoot” phenomenon. Uphill transport of thiamine under voltage-clamped conditions shows that the outwardly directed proton gradient itself acts as a driving force for thiamine uptake. Uptake of thiamine over time was next determined in the presence and absence of FCCP and valinomycin to examine the effect of pH gradient dissipation on thiamine transport (Figure 6). In the absence of ionophores, a transient intravesicular accumulation of [3H]thiamine was observed in response to an outwardly directed H+ gradient. However, in the presence of both FCCP and valinomycin, [3H]thiamine uptake was significantly decreased. These findings provide further support that an outwardly directed proton gradient is the driving force for thiamine uptake in blLPM vesicles. The presence of a transport system for thiamine in Table 2. Effect of Thiamine on pH Gradient-Dependent NMN Uptake in blLPM Vesicles NMN uptake Condition Control 1 mmol/L 2 mmol/L 5 mmol/L

thiamine thiamine thiamine

5 set 21.1 ?r3.1 22.1 + 4.8 20.7 f 2.4 17.0 f 4.0

(pmol/mg

30 set 31.2 k 27.6 + 28.8 + 25.3 f

3.8 3.1 5.2 4.0

protein)

90 min 57.8 f 8.0 55.5 * 10.1 51.1 * 13.5 54.7 k 11.6

NOTE. Membrane vesicles were preloaded in pH 5.9 media. Uptake of 50 pmol/L [3H]NMN over time was determined at 25°C in pH 7.9 media in the absence and presence of varying concentrations of thiamine. Values represent mean -t SE of quadruplicate analysis from three different membrane vesicle preparations. No significant differences were observed.

1060 MOSELEY

ET AL.

GASTROENTEROLOGY

T

-FCCP

\

I I

I’/ / 0

15s

305

80s

I

eom

Vol. 103. No. 3

ble 4, of the substrates for these organic cation/H+ antiport systems, only imipramine and choline had a significant effect on the initial rates of pH gradientdependent [3H]thiamine uptake. In particular, NMN and amiloride, as well as the thiamine analogues, oxythiamine, pyrithiamine, and amprolium, had no effect on the initial rates of pH gradient-dependent [3H]thiamine uptake. Imipramine and choline had no effect on [3H]thiamine uptake under pH equilibrated conditions and there was no significant inhibitory effect of any organic cation studied on pH gradient-dependent [3H]thiamine uptake values at equilibrium (data not shown). The presence of 1 mmol/L unlabeled thiamine inhibited pH gradientdependent [3H]thiamine uptake to values observed in the absence of a pH gradient (1.30 -t 0.06; P = 0.362). These results are consistent with a thiamine transport system with narrow substrate specificity. The liver plays a major role in the distribution and elimination of cationic drugs. Based on chemical structure, two systems have been described for the uptake of exogenous organic cations.” The hepatic transport of type I compounds, monovalent organic cations with the cationic group spatially separated

TIME Figure 4. Effect of FCCP on H’ gradient-dependent thiamine uptake in blLPM vesicles. Membrane vesicles suspended in pH 5.9 media (containing sucrose 82 mmol/L, K+ gtuconate 100 mmol/ L, MES 91 mmol/L, Tris 29 mmol/L, HEPES 14 mmol/L, Ca gluconate 0.2 mmol/L) were treated with either FCCP (20 pmol/L) or an equivalent volume of ethanol for 10 minutes at 25°C. Uptake of 1 pmol/L [3H]thiamine over time was measured in pH 7.9 media (containing sucrose 70 mmol/L, K+ gluconate 100 mmol/L, Tris 70 mmol/L, HEPES 76 mmol/L, Ca gluconate 0.2 mmol/L) at 25°C. Data expressed as mean f SE of quadruplicate analysis of three different membrane vesicle preparations. *P < 0.05; **P < 0.005.

blLPM vesicles was further substantiated by examining whether the presence of unlabeled intravesicular thiamine was capable of stimulating the uptake of extravesicular [3H]thiamine (trans-stimulation, where trans refers to the presence of the driving ion on the side opposite of the radiolabel). As shown in Table 3, in the absence of a pH gradient, initial rates of uptake of 1 mmol/L [3H]thiamine were significantly greater in blLPM vesicles preloaded with 5 mmol/L thiamine. Equilibrium uptake values were, however, similar in the presence or absence of intravesicular thiamine. The presence of an organic cation/H+ exchanger, with a broad substrate specificity, has been shown in renal, placental, intestinal, and, most recently, liver plasma membrane vesicles.‘3*‘7-26 The effects of several of these substrates on basolateral [3H]thiamine uptake in the presence of an inwardly directed pH gradient was, therefore, examined. As shown in Ta-

+VALINOMYCIN

I

0

1

//T

TIME ( minutes)

Figure 5. pH gradient-dependent thiamine uptake in presence and absence of voltage-clamped conditions in blLPM vesicles. Membrane vesicles suspended in pH 5.9 media (containing sucrose 82 mmot/L, K+ gluconate 100 mmol/L, MES 91 mmol/L, Ca gluconate 0.2 mmol/L) Tris 29 mmol/L, HEPES 14 mmol/L, were treated with valinomycin (5 pg/mg protein) or an equivalent volume of ethanol for 10 minutes at 25% Uptake of 1 pmol/ L [3H]thiamine over time was measured in pH 7.9 media (containing sucrose 70 mmol/L, K+ gluconate 100 mmol/L, Tris 70 mmol/L, HEPES 76 mmol/L, Ca gluconate 0.2 mmol/L) at 25°C. Data expressed as mean + SE of triplicate analysis of three different membrane vesicle preparations.

September

HEPATIC

1992

’ A\

I

------P +FCCP/+VALINOMYCIN

//

I

//

0

l5s

6a3

30s

TRANSPORT

1061

these exogenous organic cations on pH gradientdependent [3H]thiamine uptake in blLPM vesicles are presented. Neither PAEB (a type I compound), vecuronium (a type II compound), nor tributylmethylammonium, a cationic drug that has been considered a mixed type I and type II compound, had any significant effect on the initial rates of pH gradientdependent [3H]thiamine uptake. Previous studies have shown an amiloride-sensitive Nat/H+ exchanger on the basolateral membrane of rat liver.‘5*2*,2gAs shown above, amiloride and harmaline, which have been shown to inhibit Na+/H+ exchange in various other epithelia,30 had no effect on the initial rates of pH gradient-dependent [3H]thiamine uptake. However, to further differentiate thiamine/H+ exchange from Na’/H+ exchange, the effect of inorganic monovalent cations on [3H]thiamine uptake was examined in blLPM vesicles (Table 6). In contrast to previous reports showing inhibition of hepatic Na+/H+ exchange by lithium substitutionI pH gradient-dependent [3H]thiamine uptake was unaffected by monovalent cation substitution, except in the case of ammonium substitution, which may be acting indirectly by mediating a change in intravesicular pH by nonionic diffusion of NH,. Finally, the kinetic features of basolateral thiamine/H+ exchange were examined. The concentration dependence of the initial uptake rates was studied over a thiamine concentration range of 1 umol/-1 mmol/L. The time course of pH gradientdependent [3H]thiamine uptake was also linear for 6 seconds at high thiamine concentrations (1 mmol/L) (R2 = 0.93, y = 0.02 + 149.1X; data not shown). Carrier-mediated uptake was determined by subtracting a diffusional component of uptake ([3H]thiamine uptake under pH-equilibrated conditions) from total uptake. As shown in Figure 7, carrier-mediated uptake showed saturability with increasing concentrations of thiamine. The data in Figure 7 are presented (inset) as an Eadie-Hofstee plot (initial velocity/substrate concentration vs. initial velocity). The plot is

15

P p\p

THIAMINE

6om

TIME Figure 6. Effect of H+ gradient dissipation on thiamine uptake in blLPM vesicles. Membrane vesicles suspended in pH 5.6 media with K+ gluconate 100 mmol/L were incubated for 10 minutes at 25°C in the presence or absence of FCCP (26 pmol/L) and valinomycin (5 pg/mg protein). Control membrane vesicles were treated with equivalent amounts of ethanol. Uptake of 1 pmol/L [3H]thiamine over time was measured in pH 7.6 media with K’ gluconate 100 mmol/L at 25°C. Data expressed as mean + SE of triplicate analysis of three different membrane vesicle preparations. *P < 0.05.

from an aromatic ring structure, has typically been characterized using procainamide ethobromide (PAEB) as the model compound.z7 Vecuronium and other multivalent organic cations, in which the cationic group is masked by bulky ring structures, represent type II compounds with distinct transport characteristics.27 In Table 5, the effects of several of

Table 3. Trans-stimulation of Thiamine Uptake in blLPM Vesicles Thiamine

uptake

(nmol/mg

protein)

Condition

5 set

15 set

30sec

60 set

No thiamine,, 5 mmol/L Thiamine,,

0.30 k 0.08 0.54 IO.07"

0.53 + 0.07 0.88 + 0.11"

0.57 f 0.07 0.81 f 0.08"

0.56 2 0.11 0.72 t 0.11

60 min 1.07 + 0.08 1.04 Ik0.09

NOTE. Membrane vesicles were incubated with (in mmol/L) 250 sucrose, 100 TMA-gluconate, 50 HEPES-Tris (pH 7.5), and 0.2 Ca-gluconate in the presence or absence of 5 mmol/L thiamine. The time course of uptake of 1 mmol/L [3H]thiamine was measured in media containing (in mmol/L) 250 sucrose, 100 TMA-gluconate, 50 HEPES-Tris (pH 7.5) and 0.2 Ca-gluconate at 25°C. Extravesicular media contained 1 mmol/L thiamine at time 0. Data are means f SE of triplicate analysis of four separate membrane vesicle preparations. "P < 0.05.

MOSELEY ET AL.

1062

GASTROENTEROLOGY

Table 4. Effects of Organic Cations and Thiamine Analogues on Thiamine Uptake in blLPM Vesicles

Table 6. Effects of Inorganic Monovalent Cations on Thiamine Uptake in bJLPM Vesicles

Thiamine uptake Organic cation None Amiloride (1 mmol/L) Harmaline (1 mmol/L) N’-Methylnicotinamide (1 mmol/L) Tetraethylammonium (1 mmol/L) Imipramine (1 mmol/L) Choline (I mmoI/L) Thiamine (1 mmoI/L) Pyrithiamine (I mmol/L) Oxythiamine (1 mmol/L) Amprolium (1 mmol/L)

pmol/mg

prot/5

set

Thiamine uptake %

2.44 + 0.23 2.67 ? 0.10 2.12 * 0.19

100

2.09 * 0.15

86

2.07 1.54 1.68 1.50 2.25 2.34 2.38

85

109

a7

f 0.16 f 0.12” rfI0.10’ f 0.10” f 0.26 f 0.26 It 0.20

63 69 61 92 96 98

NOTE. Membrane vesicles were preloaded in pH 5.9 media. Five second uptake of 1 pmol/L thiamine was determined in pH 7.9 media in the absence and presence of the above organic cations and thiamine analogues. Values represent mean + SE of triplicate analysis from four different membrane vesicle preparations. “P < 0.005.

curvilinear, consistent with the involvement of two transport systems in thiamine uptake over the concentration range studied. The kinetic parameters for these transport systems were calculated by computer analysis of the data using the Michaelis-Menten equation with a nonlinear least-squares best fit, assuming two independent sites (GraphPAD Software, San Diego, CA); goodness of fit was assessed by absolute distance (R2 = 1.0). System 1 (a high-affinity, low-capacity system] showed an apparent K, of 28.6 + 12.4 pmol/L and an apparent V,,, of 36.6 & 9.6 pmol/mg prot/5 set and system 2 (a low-affinity, high-capacity system) displayed an apparent K, of

Table

5.

Effects of Type I and II Organic Cations on Thiamine Uptake in blLPM Vesicles Thiamine

Organic cation None TBuMA (1 mmoI/L) PAEB (1 mmol/L) Vecuronium (1 mmoI/L)

pmol/mg 2.17 2.37 2.59 2.09

prot/5 + 0.17 +_0.18 + 0.31 f 0.13

uptake set

Vol. 103. No. 3

% 100 109 119 96

NOTE. Membrane vesicles were preloaded in media containing (in mmol/L) 82 sucrose, 100 K+ gluconate, 91 MES, 29 Tris, 14 HEPES, and 0.2 Ca gluconate (pH 5.9). Five-second uptake of 1 pmol/L [3H]thiamine was determined in media containing (in mmol/L) 70 sucrose, 100 K+ gluconate, 70 Tris, 76 HEPES, and 0.2 Ca gluconate (pH 7.9) in the absence and presence of the above organic cations. Values represent means f SE of quadruplicate analysis from two different membrane vesicle preparations.

Inorganic cation None Sodium Potassium Lithium Rubidium Cesium Ammonium

pmol/mg 2.3 3.1 1.9 3.1 2.7 3.0 0.4

prot/5 * * * * * + f

set

0.5 0.7 0.1 0.5 0.3 0.5 O.1°

% 100 134 82 133 115 131 16

NOTE. Membrane vesicles were preloaded with pH 5.9 media (292 mmol/L sucrose, 91 mmol/L MES, 29 mmol/L Tris, 14 mmol/L HEPES, 0.2 mmol/L Ca gluconate). Five-second uptake of 1 lmol/L thiamine was determined in 270 mmol/L sucrose or 220 mmol/L sucrose plus 25 mmol/L chloride salts of the inorganic monovalent cations, buffered with 70 mmol/L Tris, 76 mmol/L HEPES, 0.2 mmol/L Ca gluconate, pH 7.9. Data are expressed as mean f SE of triplicate analysis of three different membrane preparations. “P < 0.005.

1.41 + 0.09 mmol/L and an apparent 0.05 nmol/mg prot/5 sec.

V,,,

of 1.75 *

Discussion Using vesicles derived from the basolateral (sinusoidal) plasma membrane domain of rat hepatocytes, the present study has identified and characterized an H+ gradient-dependent transport system for thiamine. First, thiamine uptake, into an osmotically active space, is enhanced in blLPM vesicles by an outwardly directed H+ gradient, resulting in a transient intravesicular accumulation of thiamine against a concentration gradient. Secondly, basolatera1 thiamine transport does not result merely from dissipation of the pH gradient, because an FCCP-induced intravesicular negative Hf diffusion potential had no additional stimulatory effect on thiamine uptake. Thirdly, under voltage-clamped conditions, uphill transport of thiamine was still observed in the presence of an outwardly directed Hf gradient. These data support the conclusion that the mechanism for H+ gradient-dependent thiamine uptake is a thiamine/H+ antiport. The presence of such a transport process is further substantiated by the ability of unlabeled intravesicular thiamine to stimulate the uptake of radiolabeled thiamine (trans-stimulation). On the basis of differences in inorganic monovalent cation sensitivitv and substrate specificity, thiamine/H+ exchange appears to be distinct from both and the recently described Na+/H+ exchange NMN+/H+ exchange. In contrast to previous findings in isolated hepato-

September

HEPATIC

1992

w

035

TliMMlNB

CONCENTRATION,

o.?a

l.0

mM

Figure 7. Kinetics of thiamine uptake in blLPM vesicles. Membrane vesicles suspended in pH 5.9 media with K+ gluconate 100 mmol/L were treated with valinomycin (5 pg/mg protein) for 10 minutes at 25°C. Five-second uptakes of varying concentrations of 13H]thiamine (1 pmol/L-1 mmol/L) were measured in pH 7.9 media with K+ gluconate 100 mmol/L at 25°C (0). A diffusional component of uptake was determined by measuring 5-second uptakes of [3H]thiamine at each substrate concentration under pH-equilibrated (pH 7.9,,.,,) conditions at 25°C (0). Carrier-mediated uptake was determined by subtracting this component from total uptake@). Straight line drawn by least squares analysis (R2 = 0.99). Data expressed as means of quadruplicate analysis of at least three different membrane vesicle preparations. Data are also given (inset) as Eadie-Hofstee (v/s vs. v) plot for carrier-mediated uptake. Straight lines represent the two carrier systems dissected by computer analysis of the experimental data. v, uptake in nmol/mg protein/5 set; s, thiamine concentration in pmol/L.

cytes showing a dependence on Na+ for thiamine transport,“3 thiamine uptake in blLPM vesicles was not enhanced by an inwardly directed Naf gradient. There are several possible explanations for this apparent discrepancy and they serve to illustrate the limitations of isolated hepatocytes as an experimental model to fully characterize membrane transport processes. Substitution of Na+ with, for example, choline or K+, and the use of ouabain and uncouplers of oxidative phosphorylation may have multiple indirect effects on hepatocellular metabolism. In fact, choline has been shown to inhibit thiamine transport in isolated rat hepatocytes31 a finding confirmed in the present study. Intracellular processes (such as metabolism and protein binding) and the loss of polarity complicate the interpretation of results in isolated hepatocytes. Intracellular events may be particularly important to control for when examining thiamine transport, because uptake appears tightly coupled to phosphorylation by thiamine pyrophosphokinase. Although thiamine transport in isolated hepatocytes can be dissociated from

THIAMINE

TRANSPORT

1063

intracellular phosphorylation, using dimethalium, a thiamine analog incapable of phosphorylation,4 thiamine accumulation after prolonged incubation has otherwise been shown to be mainly as thiamine pyrinactive, membrane ophosphate.3,4 Metabolically vesicles serve as useful models to confirm that membrane transport and intracellular trapping by phosphorylation are distinct processes in overall hepatic thiamine uptake. Our results using liver plasma membrane vesicles are similar to findings in intestinal models. Although in vivo thiamine is transported from the intestinal lumen to blood by a process that requires both Na+ and Na+,K+-ATPase activity,gx’0 inhibition of Na+, K+-ATPase activity does not prevent thiamine entry into the enterocyte or intracellular phosphorylationg Consequently, an inwardly directed Naf gradient had no effect on the transport of thiamine when examined in rat and guinea pig intestinal brush border membrane vesicles.‘1,12 Recently, thiamine transport by human erythrocytes was also shown to be a high affinity, low capacity carrier-mediated electroneutral process, which was Na+- and However, the effect of a pH energy-independent3’ gradient on thiamine transport was not examined in any of these studies. An Na+/H+ exchanger has been localized to the sinusoidal membrane in rat liver15’28,2gwhere, given the direction of the Na+ gradient, it is thought to play a role in the regulation of intracellular pH by extruding protons. Nevertheless, the resting intracellular pH in isolated and cultured rat hepatocytes averages 6.99 and 7.07, respectively,33z34 and hepatic Na+-H+ exchange, under basal conditions, has been shown to contribute little to overall Na+ uptake or H+ efflux.34 Thus, the direction of the pH gradient in the liver, under basal conditions, is most likely to be in the opposite direction, so that thiamine/H+ exchange should mediate the net uptake of thiamine into the hepatocyte. Although thiamine is a quaternary amine, thiamine/H+ exchange appears distinct from that recently described for the endogenous organic cation, NMN.13 Of the compounds shown to cis-inhibit NMN+/H+ exchange, only imipramine also inhibited thiamine/H’ exchange and thiamine had no effect on pH gradient-dependent NMN uptake. In addition, several exogenous organic cations, whose hepatic transport reportedly differs from that of NMN,35 had no effect on pH gradient-dependent thiamine uptake. Choline, which did not cis-inhibit NMN’/H’ exchange,13 inhibited pH gradient-dependent thiamine uptake. This is similar to findings in isolated hepatocytes,31 although kinetic analysis of choline uptake in the presence of thiamine indi-

1064 MOSELEY ET AL.

cated that thiamine and choline did not share common transport sites.36 Of note, in the kidney, choline has been shown to be a substrate for the brush border membrane organic cation/H+ exchanger.37 Pyrithiamine, a potent inhibitor of thiamine pyrophosphokinase, had no effect on pH gradient-dependent thiamine uptake, similar to findings in isolated hepatocytes,3 providing additional evidence that thiamine transport is distinct from phosphorylation. However, amprolium, a thiamine analogue that lacks a hydroxyethyl group, which inhibited thiamine uptake in isolated hepatocytes,3 had no effect on thiamine transport in blLPM vesicles. These findings suggest that thiamine/H+ exchange shows a high degree of substrate specificity, as might be expected for the transport of such a critical solute. The K, (28.6 ,umol/L) reported here for the highaffinity, low-capacity hepatic thiamine transport system is several orders of magnitude greater than plasma concentrations of thiamine (0.01-0.2 pmol/ may L)?38,3galthough portal thiamine concentrations exceed systemic concentrations. Thus, the physiologic relevance of the transport processes described in this study remains unclear. In isolated rat hepatocytes, two processes contribute to thiamine entry: a low-affinity process with a K, similar to that described in this study (34.1 umol/L) and a high-affinity process with a K, of 1.26pmol/L.31 However, in cells preloaded with pyrithiamine, a potent inhibitor of thiamine pyrophosphokinase, as noted above, only a single carrier system for thiamine was observed with a K, (40.5 pmol/L) similar to the low-affinity process.31 These results suggest that thiamine pyrophosphokinase, with a K, for thiamine of O.lO7.5 umol/L,3 might be involved in overall hepatic thiamine uptake by trapping intracellular thiamine in the form of thiamine pyrophosphate and thereby increasing the efficiency of thiamine uptake at physiological concentrations of thiamine. Although the liver plays a critical role in vitamin homeostasis, the mechanism for the hepatic uptake of vitamins has been studied in liver plasma membrane vesicles in only one previous study in which the transport of the water-soluble vitamin biotin was shown to be a saturable, electrogenic, Na+ gradientdependent process.4o Hepatic transport of this vitamin differed from intestinal biotin transport, as determined with brush border membrane vesicles, by its electrogenicity and inhibition by decreasing incubation buffer PH.~I In summary, the present study shows that hepatic thiamine transport, in contrast to previous results in isolated hepatocytes, appears to be dependent on a sinusoidal H+ exchange mechanism with a narrow substrate specificity that is distinct from the recently

GASTROENTEROLOGY Vol. 103,No. 3

described exchange.

organic

cation/H+

antiport

and Na+/H+

References 1. Danford

DE, Munro HN. Liver in relation to B vitamins, In: Arias IM, Jakoby WB, Popper H, Schachter D, Shafritz DA, eds. The liver: biology and pathobiology. 2nd ed. New York: Raven, 1988:505-523. 2. Chen C-P. Active transport of thiamine by freshly isolated rat hepatocytes. J Nutr Sci Vitamin01 1978;24:351-362, 3. Lumeng L, Edmondson JW, Schenker S, Li T-K. Transport and metabolism of thiamin in isolated rat hepatocytes. J Biol Chem 1979;254:7265-7268. 4. Yoshioka K, Nishimura H, Iwashima A. Active transport of dimethialium in isolated rat hepatocytes. Biochim Biophys Acta 1983;732:308-311. 5 Blitzer BL, Donovan CB. A new method for the rapid isolation of basolateral plasma membrane vesicles from rat liver: characterization, validation, and bile acid transport studies. J Biol Chem 1984;259:9295-9301. 6. Inoue M, Kinne R, Tran T, Biempica L, Arias IM. Rat liver canalicular membrane vesicles: isolation and topological characterization J Biol Chem 1983;258:5183-5188. 7 Meier PJ, Sztul ES, Reuben A, Boyer JL. Structural and functional polarity of canalicular and basolateral plasma membrane vesicles isolated in high yield from rat liver. J Cell Biol 1984;98:991-1000, 8. Meier PJ. Transport polarity of hepatocytes. Sem Liver Dis 1988;8:293-307. 9. Ferrari G, Ventura U, Rindi G. The Na+ dependence of thiamin intestinal transport in vitro. Life Sci 1971;10:67-75. 10. Hoyumpa AM, Middleton HM, Wilson FA, Schenker S. Thiamine transport across the rat intestine. I. Normal characteristics. Gastroenterology 1975;68:1218-1227. 11. Hayashi K, Yoshida S, Kawasaki T. Thiamine transport in the brush border membrane vesicles of the guinea-pig jejunum. Biochim Biophys Acta 1981;641:106-113. 12. Casirola D, Ferrari G, Gastaldi G, Patrini C, Rindi G. Transport of thiamine by brush-border membrane vesicles from rat small intestine. J Physiol 1981;398:329-339. 13. Moseley RH, Morrissette J, Johnson TR. Transport of N’-methylnicotinamide by organic cation-proton exchange in rat liver membrane vesicles. Am J Physiol 1990;259:G973-G982. 14. Meier PJ, Meier-Abt AS, Barrett C, Boyer JL. Mechanisms of taurocholate transport in canalicular and basolateral rat liver plasma membrane vesicles: evidence for an electrogenic canalicular organic anion carrier. J Biol Chem 1984;259:1061410622. 15. Moseley RH, Meier PJ, Aronson PS, Boyer JL. Na-H exchange in rat liver basolateral but not canalicular plasma membrane vesicles. Am J Physiol 1986;250:G35-G43. 16. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-275. 17. Sokol PP, Holohan PD, Grass1 PD, Ross CR. Proton-coupled organic cation transport in renal brush-border membrane vesicles. Biochim Biophys Acta 1988;940:209-218. 18. Sokol PP, Holohan PD, Ross CR. Electroneutral transport of organic cations in canine renal brush border membrane vesicles (BBMV). J Pharm Exp Ther 1985;233:694-699. 19. Takano M, Inui K-I, Okano T, Hori R. Cimetidine transport in rat renal brush border and basolateral membrane vesicles. Life Sci 1985;37:1579-1585. across brush 20. Wright SH. Transport of N’-methylnicotinamide

September

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

1992

border membrane vesicles from rabbit kidney. Am J Physiol 1985;249:F903-F911. Miyamoto Y, Canapathy V, Leibach FH. Transport of guanidine in rabbit intestinal brush-border membrane vesicles. Am J Physiol 1988;255:G85-G92. Ganapathy V. Ganapathy ME, Nair CN, Mahesh VB, Leibach FH. Evidence for an organic cation-proton antiport system in brush-border membranes isolated from the human term placenta. J Biol Chem 1988;263:4561-4568. Kinsella JL, Holohan PD, Pessah NI, Ross CR. Transport of organic ions in renal cortical luminal and antiluminal membrane vesicles. J Pharm Exp Ther 1979;209:443-450. McKinney TD, Kunnemann ME. Procainamide transport in rabbit renal cortical brush border membrane vesicles. Am J Physiol 1985;249:F532-F541. McKinney TD, Kunnemann ME. Cimetidine transport in rabbit renal cortical brush-border membrane vesicles. Am J Physiol 1987;252:F525-F535. Rafizadeh CF, Roth-Ramel F, Schali C. Tetraethylammonium transport in renal brush border membrane vesicles of the rabbit. J Pharm Exp Ther 1987;240:308-313. Meijer DKF, Mol WEM, Muller M, Kurz G. Carrier-mediated transport in the hepatic distribution and elimination of drugs, with special reference to the category of organic cations. J Pharmacokinetics Biopharm 1990;18:35-70. Arias IM, Forgac M. The sinusoidal domain of the plasma membrane of rat hepatocytes contains an amiloride-sensitive Nat/H+ antiport. J Biol Chem 1984;259:5406-5408. Fuchs R. Thalhammer T, Peterlik M, Graf J. Electrical and molecular coupling between sodium and proton fluxes in basolateral membrane vesicles of rat liver. Pfluegers Arch Eur J Physiol 1986:406:430-432. Kinsella JL, Aronson PS. Amiloride inhibition of the Na+-H+ exchanger in renal microvillus membrane vesicles, Am J Physiol 1981:241:F374-F379. Yoshioka K. Some properties of the thiamine uptake system in isolated rat hepatocytes. Biochim Biophys Acta 1984;778:201209. Casirola D, Patrini C, Ferrari G, Rindi G. Thiamin transport by human erythrocytes and ghosts. J Membrane Biol 1990; 118:11-18.

HEPATIC THIAMINE TRANSPORT

1065

33. Henderson RM. Graf J, Boyer JL. Na-H exchange regulates intracellular pH in isolated rat hepatocyte couplets. Am J Physiol 1987;252:G109-G113. 34. Renner EL, Lake JR, Persico M, Scharschmidt BF. Na+-H+ exchange activity in rat hepatocytes: role in regulation of intracellular pH. Am J Physiol 1989;256:G44-G52. 35. Steen H, Oosting R, Meijer DKF. Mechanisms for the uptake of cationic drugs by the liver: a study with tributylmethylammonium (TBuMa). J Pharm Exp Ther 1991;258:537-543. 36. Yoshioka K, Nishimura H, Himukai M, Iwashima A. The inhibitory effect of choline and other quaternary ammonium compounds on thiamine transport in isolated rat hepatocytes. Biochim Biophys Acta 1985;815:499-504. 37. Rennick BR. Renal tubule transport of organic cations. Am J Physiol 1981:240:F83-F89. 38. Burch HB, Bessey 0, Love RH, Lowry OH. The determination of thiamine and thiamine phosphates in small quantities of blood and blood cells. J Biol Chem 1952;198:477-490. 39. Weber W, Kewitz H. Determination of thiamine in human plasma and its pharmacokinetics. Eur J Clin Pharmacol 1985;28:213-219. 40. Said HM, Korchid S, Horne DW, Howard M. Transport of biotin in basolateral membrane vesicles of rat liver. Am J Physiol 1990:259:G865-G872. 41. Said HM. Redha R, Nylander W. A carrier-mediated, Na’ gradient-dependent transport for biotin in human intestinal brush-border membrane vesicles. Am J Physiol 1987; 253:G63-G636.

Received July 26, 1991. Accepted March 31, 1992. Address requests for reprints to: Richard H. Moseley, M.D., Gastroenterology Section (Ill-D), Veterans Affairs Medical Center, 2215 Fuller Road, Ann Arbor, Michigan 48105. Supported by the Medical Research Service of the Veterans Administration and by National Institute of Arthritis, Metabolism, and Digestive Diseases Grant DK39167. Part of this work was presented at the annual meeting of the American Association for the Study of Liver Diseases (AASLD), Chicago. IL, November 1990 and published in abstract form in Hepatology 11;891:1990.