Thiamine outflow from the enterocyte: a study using basolateral membrane vesicles from rat small...

Journal of Physiology (1993), 468, pp. 401-412 401With 5 figuresPrinted in Great Britain

THIAMINE OUTFLOW FROM THE ENTEROCYTE: A STUDY USINGBASOLATERAL MEMBRANE VESICLES FROM RAT SMALL INTESTINE

BY U. LAFORENZA, G. GASTALDI AND G. RINDIFrom the Institute of Human Physiology, University of Pavia, 27100 Pavia, Italy

(Received 2 July 1992)

SUMMARY

1. Rat small intestinal basolateral membrane vesicles (BLMVs) were prepared andfound to be 31 % non-vesiculated and 69% vesiculated, 49% right side out and63-8% inside out.

2. Thiamine uptake by BLMVs followed a hyperbolic time course reachingequilibrium after 60-90 min incubation. Uptake was not affected by the tra-nsmembrane potential or by the presence or absence of Na+ or K+ in the incubationmedium.

3. At concentrations below 1-25 /M, [3H]thiamine was taken up mainly by asaturable mechanism with an apparent Michaelis-Menten constant (Ki) = 1-32 ,/Mand maximal flux (Jinax) = 1-93 pmol (mg protein)-' (4 s)-1. At higher concentrations,a non-saturable mechanism prevailed.

4. Only 29% of [3H]thiamine taken up by the vesicles was membrane bound, theremaining being translocated into the vesicular space. No thiamine phosphoesterscould be detected inside the vesicles.

5. In the absence of ATP, the Na+-K+-ATPase inhibitors ouabain, frusemide andvanadate reduced thiamine uptake by 35, 30 and 15% respectively.

6. In experiments conducted with K+ inside the vesicles and Na+, Mg2+ and ATPoutside, the time course of thiamine uptake by BLMVs displayed an overshoot(80-90% increment) at 30 s incubation as compared to controls. When ATP wasreplaced with phosphocreatine, or when NaCl was replaced with isosmotic amountsof KCl, the overshoot disappeared.

7. The thiamine analogues pyrithiamine, amprolium and 4'-oxythiamine dec-reased the ATPase-dependent transport of [3H]thiamine by 100, 86 and 31%respectively.

8. These results provide evidence that the transport of thiamine by BLMVs iscoupled directly to the hydrolysis of ATP (primary active transport).

INTRODUCTION

Thiamine transport by rat everted small intestinal sacs is a Na+-dependent process(Ferrari, Ventura & Rindi, 1971; Hoyumpa, Middleton III, Wilson & Schenker,1975). However, the entry of thiamine into the enterocyte, as evaluated in brush-border membrane vesicles (BBMVs), is not influenced by Na+ (Hayashi, Yoshida &MS 1586 ) by guest on July 15, 2011jp.physoc.orgDownloaded from J Physiol (

http://jp.physoc.org/

U. LAFORENZA, G. GASTALDI AND G. RINDI

Kawasaki, 1981; Casirola, Ferrari, Gastaldi, Patrini & Rindi, 1988). Therefore theNa+-dependent mechanism of transport should be located at the contraluminal sideof the enterocyte, as suggested by findings in everted jejunal sacs (Ferrari et al. 1971;Hoyumpa et al. 1975; Hoyumpa, Nichols, Wilson & Schenker, 1977).The present investigation was aimed at characterizing the membrane mechanism

involved in the outflow of thiamine from the enterocyte. For this purpose, we usedbasolateral membrane vesicles (BLMVs) from rat small intestinal mucosa, apreparation which allows the evaluation of the membrane mechanisms of transportin the absence of metabolic or circulatory influences. Initially, we investigated thegeneral features of the basolateral uptake of thiamine. Subsequently, we assessed therole and the specificity of basolateral Na+-K+-ATPase in thiamine uptake byinvestigating the effect of different inhibitors of this enzyme, the requirements forATP and for an appropriate distribution of Na' and K+ at both sides of the plasmamembrane, and, finally, the effect of some thiamine structural analogues.The action of ATP on thiamine transport was also investigated in BBMVs from

small intestine to assess its specificity for BLMVs.A preliminary partial account of these results has been published (Gastaldi,

Laforenza, Ricci, Ferrari & Rindi, 1991; Laforenza, Casirola & Rindi, 1991).

METHODS

AnimalsAdult Wistar albino rats (200-300 g body wt) of either sex and reared on a complete standard

diet were used. The animals were killed by decapitation after 12 h fasting with water ad libitum.

Preparation of BLMVEach preparation consisted of vesicles from small intestinal mucosa obtained from six to eight

rats. All procedures were carried out at 0-4 'C. The procedure of Schron, Knickelbein, Aronson &Dobbins (1987), based on ultracentrifugation on discontinuous saccharose gradients, was used withminor modifications. Unless stated otherwise, the vesicles were washed with and suspended in amedium containing 100 mM D-mannitol, 2 mm MgSO4, 10 mm Tris-Hepes, pH 7.5 (solution A).

Purity and sidedness ofBLMVsPurity was evaluated by determining the enrichment of the activities of marker enzymes specific

for different subcellular fractions with respect to the initial mucosal homogenate. The measuredenzymes included: potassium phosphatase, for basolateral membranes (Murer, Ammann, Biber &Hopfer, 1976); saccharase, for brush-border membranes (Dahlqvist, 1964); succinate dehydro-genase, for mitochondria (Pennington, 1961); NADPH-cytochrome C reductase, for microsomes(Sottocasa, Kuylenstierna, Ernster & Bergstrand, 1967). The enrichment of basolateral membraneswas 22-2 + 0 7 times (mean + S.E.M. of 5 different preparations) as evaluated from the increment inpotassium phosphatase activity, while enrichments in saccharase, NADPH-cytochrome Creductase and succinate dehydrogenase activities were only 0-28± 0-06, 0 79± 0 05 and 0-24+ 0-07times respectively. For the evaluation ofBLMV sidedness, the content ofNa+-K+-ATPase activitywas determined according to Marin, Proverbio & Proverbio (1986) before and after treatment withsodium dodecylsulphate, in the presence and in the absence of ouabain. Na+-K+-ATPase activitywas calculated by subtracting the ouabain-insensitive ATPase activity from the total activity.Percentage and sidedness of BLMVs, calculated according to Boumendil-Podevin & Podevin(1983), were 63-8 + 8-4% inside out; 5*0 + 2-0% right side out and 31-2 + 4 0% open (sheets)(means+S.E.M. of 8 different preparations). Na+-K+-ATPase content was 1-58+0-31 ,tmolinorganic phosphate (mg protein)- min-', which is similar to previously reported values(Boumendil-Podevin & Podevin, 1983; Marin et al. 1986).

402

) by guest on July 15, 2011jp.physoc.orgDownloaded from J Physiol (


THIAMINE TRANSPORT IN MEMBRANE VESICLES

Preparation of BBMVsBBMVs were prepared from small intestinal mucosa obtained from four to five rats by using the

Ca2+ precipitation method of Kessler, Acuto, Storelli, Murer, Muller & Semenza (1978) with minormodifications. All procedures were carried out at 0-4 'C. The enrichment of brush-bordermembranes was 15-7 + 07 times (mean+ S.E.M. of 5 different preparations) as evaluated from theincrement in saccharase activity of the final preparation as compared with the initial mucosalhomogenate.

Transport efficiency of BLMVs and BBMVsTransport efficiency of the vesicular preparations was evaluated by determining the profile of D-

glucose uptake by BLMVs and BBMVs. In both cases, vesicles were incubated with 1 mM D-[U-14C]glucose (specific activity, 0-31 MBq mmol-') under the conditions described by Casirola et al.(1988).

Incubation and uptake measurementsVesicles were incubated with labelled thiamine or labelled D-glucose at 25 or 37 'C according to

different experimental conditions (see legends of figures). The amount of thiamine radioactivitytaken up by the vesicles was measured by a rapid filtration procedure (Kessler et al. 1978) usingcellulose nitrate microfilters (Microfiltration System, Dublin, CA, USA; pore diameter 0-65 or0-2 ,tm) previously saturated with unlabelled thiamine as described by Casirola et al. (1988). In eachexperiment appropriate blanks were prepared in order to evaluate the radioactivity of labelledthiamine non-specifically adsorbed on the microfilter. Blank values were subtracted from the totalradioactivity retained on the filter. Radiometric measurements were carried out by using aPackard Tri-Carb model 2000 CA liquid scintillation counter (Packard Instrument Co. Inc.,Downers Grove, IL, USA). Unless stated otherwise, all uptake values were means+S.E.M. of atleast triplicate determinations for each of five different preparations, each from six rats.

Short-time incubationsFor incubation times ranging from 4 to 15 s a STRUMA short-time incubation apparatus

(Innovativ-Labor AC, Adliswil, Switzerland) was used.

Stop solutionUnless stated otherwise, the composition of the stop solution was the following: 150 mm NaCl

and 1 mm Tris-Hepes, pH 7.5. The stop solution was used cold (0-4 °C).

Vesicular thiamine derivativesAfter incubation for 60 min with 0 5 zm [3H]thiamine in solution A plus 100 mm NaCl, BLMVs

were centrifuged for 15 min at 20000 g in the cold. The pellet, resuspended with 0 5 M HCl, was thenhomogenized and the content of thiamine and its phosphoesters determined according to theelectrophoretic micromethod of Patrini & Rindi (1980). No thiamine phosphates were producedunder these conditions.

Protein determinationProtein content was determined according to the method of Lowry, Rosebrough, Farr & Randall

(1951), using bovine serum albumin as a standard.

StatisticsThe significance of the differences of the means for different experimental conditions was

evaluated by using the following statistical methods: analysis of variance (ANOVA), followed byNewman-Keuls Q test (Snedecor & Cochran, 1967); Student's t test for paired data. All statisticaltests were carried out by using a computerized program (Glantz, 1988).

ReagentsUnlabelled thiamine chloride hydrochloride was obtained from Prodotti Roche, Milan, Italy;

pyrithiamine bromide hydrobromide (1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-hydroxy-ethyl)-2-methylpyridinium bromide monohydrobromide), 4'-oxythiamine chloride (5-(2-hydroxy-

403




ethyl)-3-[(4-hydroxy-2-methyl-5-pyrimidinyl)methyl]-4-methylthiazolium chloride), phospho-creatine (disodium salt) and Na2-ATP (vanadate free) from Sigma Chemical Co., USA; amprolium(1-[(4-amino-2-propyl-5-pyrimidinyl)methyl]-2-methylpyridinium chloride) from Merck, Sharpand Dohme, Pavia, Italy; valinomycin from Aldrich Chimica, Milan, Italy; sodium dodecylsulphate, sodium vanadate and ouabain from British Drug House (BDH) Ltd, Poole, Dorset;frusemide from Hoechst Italia, S.p.A., Milan, Italy. All other reagents were of analytical grade andsupplied by Sigma Chimica, Milan, Italy, and BDH, England.

Labelled compoundsD-[U-14C]glucose (specific activity, 9-25 MBq mmol-') and [3H]thiamine (specific activity,

429-2 GBq mmol-1) were from Amersham International plc, Amersham, England.

RESULTS

General features of thiamine uptakeTime course and Na+ effectBLMVs were incubated at 25 °C with 0-25 /aM [3H]thiamine (specific activity,

27-75 MBq mmol-1) under four experimental conditions: in the presence of an initialgradient (outward) of 100 mM-NaCl or KCl in the incubation medium; in the absenceof alkaline ions, osmotically substituted with D-mannitol; in the presence of 100 mMNaCl at both sides of the vesicle membrane, obtained by using a 20 min preincubationwith 100 mm NaCl. The incubation was stopped by adding an appropriate volume ofstop solution at fixed time intervals from 30 s to 120 min. The time course ofthiamine uptake remained virtually unmodified under all four experimentalconditions (Fig. 1).

Translocation and bindingIn order to differentiate between translocation of thiamine to the intravesicular

space and membrane binding, BLMVs were incubated at 25 'C with 0-25 ftM[3H]thiamine dissolved in media with increasing and osmometrically (Fiske OMosmometer, Fiske Associates, Burlington, MA, USA) controlled osmolarity. Whenthe values of equilibrium vesicular uptake (90 min incubation time) were plottedagainst the reciprocal of medium osmolarities, a straight line was obtained whoseintercept on the ordinate showed that 29% of thiamine take up under isosmoticconditions was membrane bound, and 71 % translocated into the vesicles (Fig. 2).

Transmembrane electrical potentialThe effect of membrane potential on the uptake of [3H]thiamine was evaluated by

imposing an electrically negative or positive potential inside the BLMVs. Twomethods were used for this purpose: (1) valinomycin-induced K+ diffusion electricalpotential, and (2) anion substitution (Said & Redha, 1988). In the first method, theuptake of 1 /M [3H]thiamine by BLMVs was evaluated in the presence ofvalinomycin, which, by causing a rapid diffusion of K+, produced a negative orpositive potential inside the vesicles depending on whether KCl was initially presentonly inside or outside the vesicles. In the second method, the uptake of 0-25 /M[3H]thiamine was measured in the presence of anions with different lipid solubility(SCN- > S042-). Incubation with the more lipid-soluble SCN- created a relatively

404




1-0.

-

._

4

E

a)

Cu

0.

CL

-c_

0.5-

0

IL0

A

IL

0

A

30 60 90

Incubation time (min)

Fig. 1. Time course of thiamine uptake by rat small intestinal basolateral membranevesicles. Thiamine uptake was measured in the presence of 0-25 /M [3H]thiamine (specificactivity, 27-75 MBq mmol-1) and: *, 100 mm NaCl (outward); 0, 100 mm NaCl, after a

20 min preincubation period; A, 100 mm KCl (outward); A, no alkaline ions, osmoticallycorrected with mannitol. Incubation medium: 100 mM D-mannitol; 2 mM MgSO4; 10 mMTris-Hepes, pH 7-5. Symbols represent means+ S.E.M. of triplicate determinations foreach of five different preparations, each from six rats. S.E.M.s were within 10% of themean values.

2-

4-WCu

Cu

0._

_)E 'ow cD

E E.' 0

=

1-

0-J0 2 4

1/medium osmolarity

Fig. 2. Effect ofmedium osmolarity on thiamine uptake by rat small intestinal basolateralmembrane vesicles. The vesicles were suspended in a solution containing 250 mMsaccharose; 2 mm MgSO4; 10 mm Tris-Hepes, pH 7-5, and incubated for 90 min at 25 °Cin media containing 0-25 ftM [3H]thiamine; 2 mM MgSO4; 10 mm Tris-Hepes, pH 7 5, andvarying amounts of saccharose in order to yield the indicated osmolarity (mosmol 1-1)(given as its reciprocal). The fitting was calculated by regression analysis (r = 0-991;P < 0-009). Number of experiments for each symbol as in Fig. 1.

405

PHY 46814 ) by guest on July 15, 2011jp.physoc.orgDownloaded from J Physiol (


406 U. LAFORENZA, G. GASTALDI AND G. RINDI

greater negative or positive intravesicular compartment (compared with the poorlylipid-soluble anion SO42-), depending on whether the anion was present outside orinside the vesicles.No statistically significant differences in [3H]thiamine uptake were observed by

using valinomycin or SCN- in comparison to controls (Fig. 3A and B).

A B A

o 06 2 290 0 4 9

0~~~~~~~~~~~

0.0,

A~~~~~~~~~~~~E0.~~~~~~~~~~~~~~~~

0 9-0 0 A-,0Incubation time (min)

Fig. 3. Effect of transmembrane potential on thiamine uptake by rat small intestinalbasolateral membrane vesicles. A, vesicles were prepared and preincubated (30 min at37 °C) in a medium containing: 2 mM MgS04; 10 mm Tris-Hepes, pH 7 5, and 100 mM D-mannitol plus 100 mm KCI (circles) or 300 mM D-mannitol (triangles). Ten microlitres ofthe vesicles were incubated at 25 °C with 200 ,l of a solution containing: 1 JiM[3H]thiamine; 50 mm NaCl; 2 mm MgSO4; 10 mm Tris-Hepes, pH 7-5; valinomycin(10lg (mg protein)-'), in the presence of: *, (intravesicular negative potential) 200 mMD-mannitol or 0 (controls) and A, (intravesicular positive potential), 100 mm KCI. B, agroup of vesicles prepared in medium containing 100 mM D-mannitol; 2 mm MgSO4;10 mM Tris-Hepes, pH 7 5, was incubated at 25 °C in the presence of 0-25 /M[3H]thiamine; 100 mM D-mannitol; 2 mm MgSO4; 10 mm Tris-Hepes, pH 75 and A,100 mM NaSCN (outward; intravesicular negative potential) or A\ 67 mM Na2SO4(outward; controls). Another group of vesicles was prepared and preincubated (30 min at37 °C) in a medium containing 100 mM D-mannitol; 2 mm MgSO4; 10 mm Tris-Hepes,pH 7-5 and 0, 100 mm NaSCN (intravesicular positive potential) or 0, 67 mM Na2SO4(controls). Thiamine uptake was measured by incubating vesicles at 25 °C in the presenceof 0-25 fM [3H]thiamine; 280 mM D-mannitol; 2 mm MgSO4; 10 mm Tris-Hepes, pH 7-5.Number of experiments for each symbol as in Fig. 1. S.E.M.s were within 10% of the meanvalues.

Kinetics of thiamine uptakeWhen BLMVs were incubated at 25 °C for 4 s with different initial concentrations

of [3H]thiamine in the presence of an initial 100 mm NaCl gradient (outward), abiphasic cumulative uptake was observed, which was non-linear at low con-centrations (approximately < 1P25 tM), and linear at higher concentrations. The bestfit of the curve, calculated by computerized least-squares regression (GraphPadSoftware, San Diego, CA, USA, 1992), could be resolved graphically into two




components according to Gastaldi, Casirola, Ferrari & Rindi (1989): a linearcomponent with expression of a non-saturable mechanism, and a hyperboliccomponent with expression of a saturable mechanism displaying Michaelis-Menten-like kinetics (Fig. 4). The apparent kinetic constants of the saturable component,

12C

e 80

0,E

E0.

4-

0. yi)

0 2-5 5 10o

Thiamine (AM)

Fig. 4. Relationship between thiamine concentration and thiamine uptake by rat smallintestinal basolateral membrane vesicles. Thiamine uptake was measured after 4 sincubation with different [3H]thiamine concentrations in the presence of an initial lO0 mmNaCl (outward) gradient. Incubation medium as in Fig. l. Symbols representmeans+ S.E.M. of at least five replicate determinations for each of seven differentpreparations. The curve of cumulative uptake and the values of the apparent Km andJmax constants of the saturable component were obtained by fitting the experimentalpoints by computerized least-squares regression. Km = 1-32 #m; Jmax = 1-93 pmol (mgprotein)-' (4 s)-1. The saturable (S) and non-saturable (N) components were obtainedgraphically from the cumulative curves (C).

calculated by computerized non-linear regression (GraphPad, 1992) were:Michaelis-Menten constant, Km = 1-32 ,ClM, and maximal flux, Jmax = 1-93 pmol (mgprotein)-' (4 s)-1.

Role of Na+-K+-A TPaseEffrect of Na+-K+-ATPase inhibitors

Three Na+-K+-ATPase inhibitors (final concentration: 2 mm for ouabain, 1-5 mmfor frusemide, 10 ,UM for vanadate) were used to investigate the involvement of thisenzyme in thiamine uptake. Ten microlitres of vesicles (prepared and preincubatedfor 15 min at 37 °C in solution A plus 10 mm KCI) were incubated for 15 s in 10 Il ofsolution A plus 0-25 /im [3H]thiamine and 100 mm NaCl, in the presence and in theabsence of a single inhibitor. Uptake decreased by 35% in the presence of ouabain,by 30% in the presence of frusemide and by 15% in the presence of vanadate (data

14-2

407




not shown). In order to assess the specificity of the inhibitory effect with respect toboth type of preparation and type of compound taken up, 2 mm ouabain was added,in separate experiments with BBMVs and BLMVs respectively, to BBMV incubationmedium containing 0-25 /M [3H]thiamine, and to BLMV incubation mediumcontaining 20 /SM [U-14C]D-glucose (incubation medium in both cases: solution A plus100 mM NaCl). In both cases, after 15 s (for BBMVs) or 4 s (for BLMVs) ofincubation, ouabain failed to affect the uptake of radioactivity. Uptake values (pmol(mg protein)-') were 0-26 + 0-07 (controls) and 0-24+ 0 03 (ouabain) for BBMVs, and8-54+ 0-74 (controls) and 9-56 + 2 16 (ouabain) for BLMVs.

Effect of ouabainBLMVs (prepared in 100mM D-mannitol; 100 mm KCl; 2 mm MgSO4; 20 mM

Tris-Hepes, pH 7 5) were incubated for 15 s at 25 °C in a medium containing: 1 ,UM[3H]thiamine; 100 mM D-mannitol; 100 mm NaCl; 2 mM MgSO4; 20 mm Tris-Hepes,pH 7-5 in the absence (control) or in the presence of 2 mm ouabain. Incubation wasstopped by adding a cooled 1-5 ml aliquot of: (i) isotonic solution, (ii) hypotonicsolution, or (iii) hypotonic solution containing 2 mm ouabain. After 5 min, theBLMVs or the basolateral membrane sheets produced by the hypotonic solution werefiltered on cellulose nitrate microfilters (pore diameter 0-2 /Lm) and the relevantradioactivity measured. Ouabain inhibited (35%) [3H]thiamine uptake (pmol (mgprotein)-1) to the same extent for both BLMVs ((i): controls, 0-86+0-12; ouabain,0-55+ 0-05) and membrane sheets ((ii): controls, 0-92 + 016; ouabain, 0-60+ 015).Moreover, the presence of ouabain in the hypotonic stop solution caused a furtherinhibition (62%) of thiamine uptake ((iii): controls, 0-34 + 013; ouabain, 0-13 + 0-07)by sheets incubated with or without ouabain.

Effect ofATP on BLMV uptakeWashed BLMVs were resuspended in the appropriate solution and incubated at

37 °C after adding nineteen volumes of incubation medium (see legend of Fig. 5)containing 1 /,M [3H]thiamine and 5 mm Na2-ATP (ATP) or 5 mm Na2SO4 (controls) toone volume of vesicle suspension. The incubation was stopped at time intervalsranging from 15 s to 30 min. Valinomycin was added in order to facilitate inflow ofK+into BLMVs. Under these conditions (presence of ATP, and Na+, K+ and Mg2+appropriately distributed), which are known to be associated with efficient Na+-K+-ATPase activity, [3H]thiamine uptake rate showed an overshoot, with anapproximately 85% increment compared to controls (absence of ATP) (Fig. 5A).The overshoot was statistically significant (P < 0-01) at both 30 and 60 s incubationtimes and disappeared when ATP was substituted with phosphocreatine (disodiumsalt (Fig. 5 C) or when Na+ was substituted with K+ (Fig. 5D). When BBMVs insteadof BLMVs were similarly incubated, no overshoot was observed (Fig. 5B).

Structural specificity ofA TP-dependent uptakeThe potency of some structural analogues of thiamine (pyrithiamine, amprolium

and 4'-oxythiamine) and choline in inhibiting the Na+-K+-ATPase-dependentovershoot of [3H]thiamine uptake was evaluated by incubating BLMVs for 30 s withand without ATP (for details see legend of Fig. 5). Choline and analogues of thiamine

408



THIAMINE TRANSPORT IN MEMBRANE VESICLES 409

were added to the incubation medium at an initial concentration of 10/M([analogue]:[3H]thiamine] = 10). Choline had no effect, whereas amprolium, pyr-ithiamine and 4'-oxythiamine significantly decreased [3H]thiamine uptake by 100, 86and 31 %, respectively.

B8, A

6

4

201

8-

6

4.

2

4-

2-

0-

c0 1 3 10 30

8,D

6-

0 1 32-

10 30 01 3 10 30

Incubation time (min)

Fig. 5. Specificity of the Na+-K+-ATPase-dependent transport of thiamine by rat smallintestinal basolateral vesicles. A (basolateral membrane vesicles) and B (brush-bordermembrane vesicles): 10lO of vesicles suspended in 80 mM D-mannitol; 100 mm KCI;10 mM Tris-Hepes, pH 7-5; valinomycin (10 ,ug (mg protein)-'), were incubated at 37 °Cwith 190 /1 of a medium containing: 1 /UM [3H]thiamine; 55 mM D-mannitol; 100 mM

NaCl; 5 mM MgSO4; 10 mm Tris-Hepes, pH 7 5, in the presence of 5 mm Na2-ATP (@) or

5 mM Na2SO4 (control; 0). In C andD the experimental conditions were as in A, but Na2-ATP was substituted with phophocreatine (disodium salt) (C) or NaCl was substitutedwith isosmotic KCI (D). Control (0) was Na2SO4. Symbols represent means+ S.E.M. of fivereplicate determinations for each of five different preparations. *P < 0-01 vs. control(Student's t test).

DISCUSSION

The purity and the functional efficiency of BLMV and BBMV preparations,evaluated from the time course of D-glucose transport (data not shown), were

comparable to those reported in the literature and considered suitable for transport

0._

4-1

0

E

E0.a)

0.

CL

CU

I-M




studies (Del Castillo & Robinson, 1982; Kikuchi & Ghishan, 1987; Said, Redha &Nylander, 1988). In BLMVs the vesicular membrane was on average 64% inside out,a percentage similar to that reported for both human and guinea-pig intestine (DelCastillo & Robinson, 1982; Kikuchi & Ghishan, 1987; Said et al. 1988).When ATP was absent in the incubation medium, the time course profile of

[3H]thiamine uptake by BLMVs was virtually identical in the presence and in theabsence of Na+ (Fig. 1), a finding similar to that reported for BBMVs (Hayashi et al.1981; Casirola et al. 1988). This suggests that, in the absence of ATP, Na+ was notinvolved in thiamine outflow from the enterocyte. However, Na+ was important forthe full activation of Na+-K+-ATPase when ATP was present in the medium andwhen the active mechanism of thiamine transport was operating (see below).

Experiments with media of increasing osmolarity showed that translocation wasthe main mechanism of [3H]thiamine uptake by BLMVs (Fig. 2): only 29 % of thevitamin was membrane bound, an amount much higher than that reported forBBMVs, where binding is virtually negligible (Casirola et al. 1988). Translocation wasnot associated with thiamine phosphorylation.Although thiamine dissociates as a monovalent cation at pH 7.5 (Komai & Shindo,

1974), uptake was not affected by a relatively negative charge in the intravesicularspace (Fig. 3A and B), suggesting that transport is probably an electroneutralprocess, as also reported for intact intestinal tissue (Hoyumpa et al. 1975), BBMVs(U. Laforenza & G. Rindi, unpublished data) and erythrocytes and ghosts (Casirola,Patrini, Ferrari & Rindi, 1990). It is noteworthy that a relatively positive charge inthe intravesicular space, corresponding to the external side of the plasma membraneof the intact enterocyte, also failed to influence thiamine uptake by BLMVs. Sinceour vesicle preparations were mainly inside-out oriented, this finding suggests thatboth the normal potential across the basolateral membrane of the intact cell and thepotential possibly generated in vitro by Na+-K+-ATPase activity (see below) areunable to induce thiamine translocation.At concentrations below 1-25 saM, [3H]thiamine was taken up into BMLVs by a

saturable (Michaelis-Menten) process (Fig. 4). Usually the physiological intraluminarconcentration of thiamine is lower than 2 /tM in both man and rat (Hoyumpa et al.1975; Sklan & Trostler, 1977). Therefore, the intestinal transport of thiamineappears to be accounted for predominantly by a saturable mechanism at both theluminal (Casirola et al. 1988) and contraluminal sides of the enterocyte. Since theJmax value of thiamine uptake by BLMVs is 6-fold greater than that recorded inBBMVs (Casirola et al. 1988), outflow from the enterocyte is not a rate-limitingprocess in thiamine absorption.

Different inhibitors ofNa+-K+-ATPase activity effectively inhibited [3H]thiamineuptake by BLMVs. The inhibition caused by ouabain was specific for both BLMVs(thiamine uptake in BBMVs was unaffected) and thiamine (uptake of D-glucosein BLMVs was unaffected). Inhibition by ouabain involved displacement of[3H]thiamine from its binding sites on the basolateral plasma membrane, assuggested by the results obtained with membrane sheets. Thus, the much greater[3H]thiamine binding capacity of BLMVs as compared to BBMVs may be ascribedto the almost exclusive presence in BLMVs of Na+-ATPase, to the externalconfiguration of which ouabain can bind.

410



THIAMINE TRANSPORT IN MEMBRAANE VESICLES

The inhibitory effect of frusemide could explain the recent finding that long-termadministration of frusemide may induce thiamine deficiency both in rats (Yui,Itokawa & Kawai, 1980) and in humans (Seligmann et al. 1991). By inhibitingNa+-K+-ATPase, frusemide may reduce thiamine transport in the kidney and in theintestine and prevent, in this way, both the retention and the uptake of the vitamin,with consequent depletion of its body stores.The observation that ouabain inhibited thiamine uptake by BLMVs is in

agreement with data of previous studies, in which ouabain was found to reducethiamine transport in intact small intestinal tissue (Ferrari et al. 1971; Hoyumpa etal. 1975) and in isolated hepatocytes (Lumeng, Edmondson, Schenker & Li, 1979;Yoshioka, 1984). Under experimental conditions associated with complete activationof Na+-K+-ATPase in the intact tissue, BLMVs were able to actively transportthiamine as indicated by the overshoot shown in Fig. 5A. The overshoot disappearedafter altering the distribution of ions or the type of high energy phosphate (Fig. 5Dand C), indicating that thiamine transport was strictly dependent on functionalNa+-K+-ATPase. Since a positive charge inside the vesicle did not by itself influencethiamine transport (Fig. 3), these findings provide the first direct evidence for aninvolvement of Na+-K+-ATPase in the intestinal transport of thiamine. Based on theresults of inhibition experiments with thiamine analogues, this mechanism appearsto be specific for thiamine. Moreover, the same mechanism may account not only forthiamine transport against a concentration gradient in intact intestinal tissue butalso for its overall Na+ dependence, since Na+ is not required for the passage of thevitamin across the brush-border membrane.

This research was supported by grants from Italian MPI (40 %; 1987) and MURST (60 %; 1991).We wish to express our gratitude to Professor E. Perucca (Clinical Pharmacology Unit, Universityof Pavia, Pavia, Italy) for revising the English, and to the following firms for generous gifts ofthiamine and analogues: Prodotti Roche, Milan, Italy; Merck, Sharp and Dohme, Pavia, Italy. Thesecretarial assistance and excellent typing of Mrs P. Vai Gatti and Mrs M. Agrati Greco aregratefully acknowledged.

REFERENCES

BOUMENDIL-PODEVIN, L. F. & PODEVIN, R. A. (1983). Isolation of basolateral and brush-bordermembranes from the rabbit kidney cortex. Vesicle integrity and membrane sidedness of thebasolateral fraction. Biochimica et Biophysica Acta 735, 86-94.

CASIROLA, D., FERRARI, G., GASTALDI, G., PATRINI, C. & RINDI, G. (1988). Transport of thiamineby brush-border membrane vesicles from rat small intestine. Journal ofPhysiology 398, 329-339.

CASIROLA, D., PATRINI, C., FERRARI, G. & RINDI, G. (1990). Thiamin transport by humanerythrocytes and ghosts. The Journal of Membrane Biology 118, 11-18.

DAHLQVIST, A. (1964). Method for assay of intestinal disaccharides. Analytical Biochemistry 7,18-25.

DEL CASTILLO, J. R. & ROBINSON, J. W. L. (1982). The simultaneous preparation of basolateraland brush-border membrane vesicles from guinea-pig intestinal epithelium, and the de-termination of the orientation of the basolateral vesicles. Biochimica et Biophysica Acta 688,45-56.

FERRARI, G., VENTURA, U. & RINDI, G. (1971). The Na+-dependence of thiamin intestinal transportin vitro. Life Sciences 10, 67-75.

GASTALDI, G., CASIROLA, D., FERRARI, G. & RINDI, G. (1989). Effect of chronic ethanoladministration on thiamine transport in microvillous vesicles of rat small intestine. Alcohol andAlcoholism 24, 83-89.

411




GASTALDI, G., LAFORENZA, U., Ricci, V., FERRARI, G. & RINDI, G. (1991). Thiamin transport inbasolateral membrane vesicles from rat small intestine: preliminary observations. PfluigersArchiv 419, R27.

GLANTZ, S. A. (1988). Statistica per Discipline Biomediche: Programma Applicativo. McGraw-HillLibri Italia, Milano.

HAYASHI, K., YOSHIDA, S. & KAWASAKI, T. (1981). Thiamine transport in the brush bordermembrane vesicles of the guinea pig jejunum. Biochimica et Biophysica Acta 641, 106-113.

HOYUMPA, A. M. JR, MIDDLETON III, H. M., WILSON, F. A. & SCHENKER, S. (1975). Thiaminetransport across the rat intestine. I. Normal characteristics. Gastroenterology 68, 1218-1227.

HOYUMPA, A. M. JR, NICHOLS, S. G., WILSON, F. A. & SCHENKER, S. (1977). Effect of ethanol onintestinal (Na, K) ATPase and intestinal thiamine transport in rats. The Journal of Laboratoryand Clinical Medicine 90, 1086-1095.

KESSLER, M., AcUTO, D., STORELLI, C., MURER, H., MULLER, M. & SEMENZA, G. (1978). A modifiedprocedure for the rapid preparation of efficiently transporting vesicles from small intestinalbrush border membranes. Biochimica et Biophysica Acta 506, 136-154.

KIKUCHI, K. & GHISHAN, F. K. (1987). Phosphate transport by basolateral plasma membranes ofhuman small intestine. Gastroenterology 93, 106-113.

KOMAI, T. & SHINDO, H. (1974). Structural specificities for the active transport system of thiaminein rat small intestine. Journal of Nutritional Science and Vitaminology 20, 179-187.

LAFORENZA, U., CASIROLA, D. & RINDI, G. (1991). Na/K ATPase is involved in the transport ofthiamin by basolateral membrane vesicles from rat small intestine. First International Congresson 'Vitamins and Biofactors in Life Science', Kobe (Japan) Abstracts, p. 116.

LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L. & RANDALL, R. J. (1951). Protein measurementwith the Folin phenol reagent. Journal of Biological Chemistry 193, 265-275.

LUMENG, L., EDMONDSON, J. W., SCHENKER, S. & LI, T. K. (1979). Transport and metabolism ofthiamine in isolated rat hepatocytes. Journal of Biological Chemistry 254, 7265-7268.

MARIN, R., PROVERBIO, T. & PROVERBIO, F. (1986). Inside-out basolateral plasma membranevesicles from rat kidney proximal tubular cells. Biochimica et Biophysica Acta 858, 195-201.

MURER, H., AMMANN, E., BIBER, J. & HOPFER, U. (1976). The surface membrane of the smallintestinal epithelial cell. I. Localization of adenyl cyclase. Biochimica et Biophysica Acta 433,509-519.

PATRINI, C. & RINDI, G. (1980). An improved method for the electrophoretic separation andfluorometric determination of thiamine and its phosphates in animal tissues. InternationalJournal for Vitamin and Nutrition Research 50, 10-18.

PENNINGTON, R. J. (1961). Biochemistry of dystrophic muscle. Mitochondrial succinate-te-trazolium reductase and adenosine triphosphatase. Biochemical Journal 80, 649-654.

SAID, H. M. & REDHA, R. (1988). Biotin transport in rat intestinal brush-border membranevesicles. Biochimica et Biophysica Acta 945, 195-201.

SAID, H. M., REDHA, R. & NYLANDER, W. (1988). Biotin transport in basolateral membranevesicles of human intestine. Gastroenterology 94, 1157-1163.

SCHRON, C. M., KNICKELBEIN, R. G., ARONSON, P. S. & DOBBINS, J. W. (1987). Evidence forcarrier-mediated Cl-S04 exchange in rabbit ileal basolateral membrane vesicles. AmericanJournal of Physiology 253, G404-410.

SELIGMANN, H., HALKIN, H., RAUCHFLEISCH, S., KAUFMANN, N., TAL, R., MOTRO, M., VERED, Z.& EZRA, D. (1991). Thiamine deficiency in patients with congestive heart failure receiving long-term furosemide therapy: a pilot study. American Journal of Medicine 91, 151-155.

SKLAN, D. & TROSLTER, N. (1977). Site and extent of thiamin absorption in the rat. Journal ofNutrition 107, 353-356.

SNEDECOR, G. W. & COCHRAN, W. G. (1967). Statistical Methods, 6th edn., pp. 258-298. Iowa StateUniversity Press, Ames, 10, USA.

SOTTOCASA, G. L., KUYLENSTIERNA, B., ERNSTER, L. & BERGSTRAND, A. (1967). An electron-transport system associated with the outer membrane of liver mitochondria. A biochemical andmorphological study. Journal of Cell Biology 32, 415-438.

YOSHIOKA, K. (1984). Some properties of the thiamine uptake system in isolated rat hepatocytes.Biochimica et Biophysica Acta 778, 201-209.

YUI, Y., ITOKAWA, Y. & KAWAI, C. (1980). Furosemide-induced thiamine deficiency. CardiovascularResearch 14, 537-540.

412



Thiamine outflow from the enterocyte: a study using basolateral membrane vesicles from rat small...

Documents

Transcript of Thiamine outflow from the enterocyte: a study using basolateral membrane vesicles from rat small...