Pfliigers Archiv
Pfl~gers Arch. 373, 243-248 (1978)
EuropeanJournal of Physiology 9 by Springer-Verlag 1978
Sugar Transport by Brush Border Membrane Vesicles Isolated from Human Small Intestine H. LUCKE, W. BERNER*, H. MENGE**, and H. MURER Max-Planck-Institut f/Jr Biophysik, Kennedyallee 70, D-6000 Frankfurt (Main), Federal Republic of Germany
Abstract. Uptake of D- and L-glucose, and fructose by purified brush border membrane vesicles isolated from human small intestine was studied using a rapid filtration technique. The uptake of D-glucose by the vesicles was osmotically sensitive and represented transport into an intravesicular space and not binding to the membranes. Transport of both, D- and L-glucose was inhibited by phlorizin. Uptake of D-fructose in~Lothe brush border vesicles was not stimulated by sodium. In the presence of a sodium gradient D-glucose was taken up 5 times faster than L-glucose. The amount of D-glucose transported into the vesicles in the presence of a sodium gradient was transiently higher than the amount of D-glucose taken up at equilibrium (overshoot). D-Glucose transport was stimulated only by a sodium gradient; other monovalent cations had no effect. In the presence of a sodium gradient D-glucose transport was increased by the lipophilic anion thiocyanate and decreased by the nearly impermeable anion sulfate as compared with uptake of D-glucose in the presence of a sodium chloride gradient. This indicates an influence of the electrical membrane potential on the sodimn coupled non-electrolyte transport. Key words: Man - Intestine - Brush border - Sugar - Transport.
INTRODUCTION In the "sodium gradient hypothesis" Crane postulated that a molecular coupling between sodium and glucose flux in the brush border membrane was responsible for * From the Department of Surgery, Uni'~ersityHospital, D-3000 Hannover, Federal Republic of Germany ** From the Department of Medicine, University Hospital, D_3550 Marburg/Lahn, Federal Republic of Germany
intestinal active sugar transport [4, 5]. However, it was difficult to verify the physiological significance of this mechanism by in vivo perfusion experiments using laboratory animals [7,9] and man [21,22]. The differences in the interpretation of the results obtained by studies with the intact epithelial tissue in vivo can be explained by methodological difficulties in controlling different parameters such as paracellular fluxes and cellular metabolism. In particular, large unstirred layers make it almost impossible to determine substrate and cosubstrate concentrations at the transport sites itself [6]. It is therefore very difficult to get data from in vivo perfusion experiments which allow definition of the driving forces of a Na+-gradient on Na + coupled transport processes. In the last few years methods have been developed to study directly the role of the brush border membrane in reabsorptive processes. Glucose, fructose, amino acid, inorganic phosphate and sodium transport systems have been demonstrated in vesiculated brush border membranes isolated from the small intestine of rats [2, 12, 13,19,25,26]. It has been shown, that the transport of glucose, amino acids and inorganic phosphate is stimulated by sodium via flux coupling of the cosubstrate sodium with the different substrates. Similar experiments were also carried out with brush border membrane preparations isolated from renal proximal tubule [8, 14]. The aim of the present contribution is to explore whether these well documented mechanisms of sodium dependent non-electrolyte movement play also a role in human intestinal glucose absorption. For that we have undertaken the study of glucose transport in brush border membrane vesicles isolated from human small intestine. In such preparations the influence of cellular metabolism is avoided, and since the composition of intra- and extracellular fluid can be easily manipulated, the driving forces for glucose can be defined.
0031-6768/78/0373/0243/$ 01.20
244
The data reported in this contribution show the presence of a cotransport system for sodium and Dglucose (and L-glucose) in the human brush border membrane similar to that described in brush border membranes isolated from laboratory animals.
Pfltigers Arch. 373 (1978) Vibrated epithelial cell suspension
METHODS AND MATERIALS Isolation of Brush Border Membrane Vesicles. 25 cm long pieces of histologically normal human upper jejunum, which were obtained from 2 patients in the course of surgery for tumors involving other tissues of gastrointestinal tract, were rinsed with Ringer solution and kept frozen at minus 40~ C. For membrane isolation 20 g of the frozen material was thawed in Ringer solution at room temperature. The muscularis mucosae was separated from the mucosal layer by dissection. The dissected mucosal pieces were placed in 60 ml of mannitol buffer (300 mM mannitol, 12mM Tris-HC1, pH 7.1) and disrupted in a vibro-blendor (Chemap AG, M/innedorf/ZH, Switzerland). The suspension obtained by vibration of the mucosal pieces was filtered through a nylon sieve (pore size 0.8ram). As indicated by phase contrast microscopy the filtered suspension contained mainly epithelial cells, brush border fragments and free nuclei. This suspension of epithelial cells was homogenized at 0 ~ C in hypotonic medium as described in scheme 1. Membrane isolation was performed by a modification of the procedure of Schmitz et al. [23] as indicated in scheme 11 All the centrifugation steps were carried out in a Sorvall RC 2B refrigerated centrifuge (rotor SS34).
9 discard pellet 1 Supernatant 1
Transport Studies
centrifuge at 20000g for 30min ~discard supernatant 2
Pellet 2
resuspend in 70 ml of mannitol buffer used for transport studies (100 mM mannitol, 20 mM HEPES2-Tris, pH7.4) with a glass teflon homogenizer (10 strokes at 1400rpm); add CaCIz to a final concentration of 10 mM; after 15 min centrifuge at 7500 g for 15 min ,discard pellet 3
Supe}natant 3
centrifuge at 20000 g for 25 min discard supernatant 4
Pellet 4
Protein Concentrations and Enzyme Activities Protein determination was carried out according to Lowry et al. [15] with bovine serum albumin as standard (Behring-Werke, Marburg, W.-Germany). Alkaline phosphatase (EC 3.1.3.1.), an enzyme marker for the intestinal brush border membrane was measured as described by Berner et al. [1]. K+-stimulated pnitrophenyl-phosphatase (EC 3.1.3.41), a marker of the contraluminal plasma membrane, was assayed as reported by Murer et al. [18].
after addition of 240 ml icecold bidest H20 to 60 ml of the vibrated epithelial cell suspension (in a buffer containing 300raM mannitol, 12 mM Tris-HC1, pH 7.1) homogenize with an Universal blendor (MX 32; Braun GmbH,. Melsungen, W.-Germany), 3.5rain at top speed; add CaC12 to a final concentration of 10raM; after 15min centrifuge at 7500g for 15 min
resuspend in 2ml of mannitol buffer (as above) by sucking the suspension 7 times through a stainless steel needle (0.9 x 38 mm) into a plastic syringe; centrifuge at 2600 g for 5 min discard pellet 5
+
Supernatant 5
centrifuge at 15000g for 20min in an Eppendorf table centrifuge (3200) discard supernatant 6
Pellet 6
final brush border vesicle preparation
Scheme 1. Preparation of brush border membrane vesicles from small intestinal epithelial cells
Uptake of labelled compounds by isolated brush border membrane vesicles were measured by a Millipore filtration technique as described previously [13,8]. The exact compositions of the incubation media are given in the legends to the figures.
Materials The labelled compounds were purchased from New England Nuclear Corp. (Boston, MA, U.S.A.). All other chemicals used in the experiments were obtained from Merck (Darmstadt, W.-Germany) with the exceptions of HEPES 2 (Serva, Heidelberg, W.-Germany) and phlorizin (Roth, Karlsruhe, W.-Germany). All chemicals were of the highest purity available. 1 A similar isolation procedure was developed recently for the isolatiotl of brush border membranes from rabbit and rat small intestine (Kessler, M., Acuto, O., Storelli, C., Murer, H., Mfiller, M., Semenza, G. : Biochim. Biophys. Acta, submitted for publication) 2 HEPES: N-2-hydroxyethylpiperazine-N'-ethanesulphonic acid
RESULTS
A. Purity of Brush Border Membrane Vesicles The specific activity of the brush border membrane m a r k e r e n z y m e , a l k a l i n e p h o s p h a t a s e [12,23], w a s 11 t i m e s h i g h e r i n t h e f i n a l m e m b r a n e f r a c t i o n as c o m p a r e d w i t h t h e s t a r t i n g h o m o g e n a t e ( s h o w n i n T a b l e 1). K+-stimulated p-nitrophenyl-phosphatase was only slightly enriched in the purified brush border memb r a n e ( 1 . 6 - f o l d ) , s u g g e s t i n g little c o n t a m i n a t i o n b y b a s a l - l a t e r a l m e m b r a n e s [12].
245
H, Liicke et al. : Human Intestinal Sugar Transport Table 1. Enrichment of marker enzymes in the brush border vesicle preparation MW + S.D. n = 4 Total homogenate (mU/mg of protein)
Vesicle fraction (mU/mg of protein)
Enrichment factor
106.58 + 31.64 4.82 • 5.42
1196.65 +_ 444,83 5.81 +_ 4.69
11.16 _+ 1.79 1.61 • 0.64
Alkaline Phosphatase K+-stimulatedp-nitrophenyl-phosphatase
9 NaSCN gradient
~
I
oNoSCN gradient t D-glucose uptake + 0.SmM phlorizin 9 KSCN gradient § 22NG
e NaSCN gradient
~ 120._ ~_
9 K SCN gradient
~-
uptake
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,
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Incubation time [min]
120
o Na SCN gradient o NQSCN gradient
-
+0 5 cam phlorizin
~
~g O
--o--
80-
1
2 Incubation time [mini
60
Fig. 2. Time course of D-fructose uptake by brush border membranes. The vesicles were loaded with a buffer containing 100 mM mannitol and 20raM HEPES/Tris (pH7.4) and incubated in a medium containing 0.1mM (3H) fructose and 100raM NaSCN (E]) or 100mM KSCN ( I ) , respectively. Fructose uptake is expressed as percentage of the amount taken up by the vesicles after 60 min of incubation time in the NaSCN (1:5) medium, which amounted to 234 pmoles/mg of protein
~
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p~
J
--A--
9 KSCN gradient
121_221
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1 2 fncubcltion time [mi,q]
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Fig. I. Time course of D-, L-glucose and 2ZNa uptake by human intestinal brush border vesicles. Membrane vesicles isolated according to scheme 1 and loaded with a buffer containing 100mM mannitol and 20 m M HEPES/Tris (pH 7.4) were incubated at 25 ~ C in a medium containing 0.1 m M D-(Ir (a) or 0.1raM L(3H)glucose (b), 100 m M mannitol, 20ram HEPES/Tris (pH 7.4), 100ram NaSCN (O) or 100raM Z2NaSCN ( + ) or i 0 0 m M NaSCN plus 0.5 mM pMorizin (O) or 100 m M KSCN ( , ) . Glucose uptake is expressed as percentage o f the amount taken up by the membranes after 60 min of incubation in the NaSCN (O) medium. The absolute value for the NaSCN (e) medium was 241 pmoles/mg of protein (a) and 204pmoles/mg of protein (b) after 60 min of incubation, for 22NaSCN ( + ) it was II7nmoles/mg of protein after 60min of incubation
B. Transport Properties When the two stereoisomers D- and L-glucose were added to the isolated brush border vesicles, different time courses of uptake were obtained (Fig. 1a, b). The amount of D-glucose taken up by the vesicles in the presence of a sodium gradient during the first 60 s of
incubation was about 4.6 times higher than that of eglucose. This difference was reduced by about half if sodium was replaced by potassium. In the presence of a NaSCN gradient D-glucose uptake showed an "overshoot" phenomenon, during which the intravesicular glucose concentration transiently exceeded the concentration of glucose in the incubation medium. The "overshoot" arose because of the persistance of a sodium gradient when the intravesicular glucose had already reached the concentration of the incubation medium. D-Glucose concentration within the vesicles reached the concentration of the medium in less than 10 s, whereas sodium reached only 30 ~ of equilibrium after this incubation period (Fig. 1a). The initial uptake olDglucose was stimulated 3.6 times in the presence of a sodium gradient compared with the presence of a potassium gradient. L-Glucose uptake was only slightly stimulated by sodium (1.5-fold). D-Fructose, which is thought from experiments with animal preparations to be transported by sodium-independent "facilitated diffusion" [25], showed also no sodium stimulated transport in human intestinal brush border vesicles (Fig. 2).
246
Pfltigers Arch. 373 (1978)
//
~_ 80-
60-
o D-gLucose T z; D-glucose plus O5 mM ph[orizin -t~ 9 D-gtucose(rninus O-glucose with 9hlorizin) i
-.~
t
~6
./ /
/.0i
20/ /
/ t
/
-~cL 2000
9
o
t
/ / ~-
1000
& 0 0
i
i
i
i
I
I
i
1
2
3
4
5
6
7
Osmo[flrity
0
-1
1
2 3 D- glucose rmM]
Z~
5
Fig. 3. Influence of medium osmolarity on D-glucose uptake by isolated brush border membrane vesicles. The uptake of 0.1 mM D(l~C)glucose was determined in the presence of I00 mM mannito[, 20 mM HEPES/Tris (pH 7.4), t0 mM NaCI and sufficientcetlobiose to give the indicated osmolarity. The values given represent equilibrium values obtained after 15min of incubation
Fig.4. Influenceof D-glucoseconcentration on D-glucoseuptake by brush border membrane vesicles. The incubation medium contained 100mM mannitol, 20 mM HEPES/Tris (pH 7.4), 100mM NaSCN and D-glucose (O), or D-glucose plus 0.5 mM phlorizin (A) at the indicated concentrations(Fig.4a). The specificsodium dependent Dglucose uptake ([3) is obtained by the total uptake (O) minus the phlorizin inhibited part (A). The amounttaken up by the vesiclesafter 60 s is given
The amount of D-glucose taken up by the vesicles at equilibrium was in inverse proportion to the osmolarity of the incubation medium (Fig.3). These findings indicate the presence of osmotically reactive membrane vesicles and secondly transport of glucose into an intravesicular space. Such findings cannot be explained by binding of substrates to or incorporation of substrates into the membrane. The amount of o- and L-glucose taken up after 60 s into brush border membrane vesicles increased in curvilinear fashion when the glucose concentration in the incubation medium was increased (Fig. 4). In the presence of phlorizin, which inhibits the sodium dependent glucose transport [5,27], a smaller but linear Dglucose uptake was observed which might indicate uptake by simple diffusion. The carrier mediated sodium dependent o-glucose transport is supposed to be the difference of the total uptake minus the phlorizin inhibited part of o-glucose uptake. In the concentration range used in this experiment (Fig.4) no complete saturation of o-glucose was observed. This indicates that the apparent Michaelis constant of the transport system (Kin) must be rather high (above 5 mM). This low affinity of the transport system for glucose is further supported by kinetic analysis (data not shown) of the initial (15 s) uptake rates (Lticke, H., Berner, W., Menge, H., Murer, H. : unpublished results). L-Glucose uptake was also inhibited by phlorizin as it was slightly stimulated by sodium (Fig. 1 b). From
Table 2. Effect of cations on D-glucoseuptake into intestinal brush border membrane vesicles (amount taken up during the first 60s). The experiments were carried out in an incubation mediumcontaining 100raM mannitol, 20mM HEPES/Tris (pH7.4), 0.1 mM D(14C)glucose and different salt gradients as given in the table Salt in the incubation medium
D-Glucose uptake
(0.1 M)
(pmoles/mg of protein)
Choline chloride LiC1 NaC1 KCI RbC1 CsCI
43.5 43.7 114.5 54.4 41.1 40.9
these experiments the existence of carrier mediated Lglucose uptake could be inferred [3,12,20]. Table 2 shows the effect of various cations on oglucose transport by brush border membrane vesicles. The highest stimulation was seen with sodium. Among the other cations tested no stimulatory effect could be detected as compared with the uptake in the presence of a choline gradient. As shown in Table 3 replacement of CI- in the presence of a sodium gradient by a more permeant anion such as S C N - stimulated the initial uptake of glucose. As we have demonstrated earlier in experiments with rat small intestinal preparations this
H. Liicke et al. : Human Intestinal Sugar Transport Table3. Effect of anion replacement on D-glucose uptake into intestinal brush border membrane vesicles (amount taken up during the first 60 s). The incubation medium contained 100 mM mannitol, 20 mM HEPES/Tris (pH 7.4), 0.1 mM D-(l~C)glucose and different salt gradients as indicated in the table Salt in the incubation medium
D-Glucose uptake (pmoles/mg of protein)
0.1 M NaSCN 0.1M NaC1 0.05 M Na2SO 4 + 0.05 M mannitol
224.7 114.5 94.4
effect is best explained by the assumption of a hyperpolarisation (vesicle inside negative) of the membrane potential in the presence of a SCN- gradient [8, 16]. If the membrane potential is reversed by the use of SO l instead of C1-, in the presence of a sodium gradient the rate of uptake is markedly reduced. These results strongly point to an involvement of the membrane potential in the sodium dependent transmembranal movement of glucose and to a rheogenicity of the flux coupling mechanism between sodium and glucose.
DISCUSSION Our studies on the D-glucose transport by brush border vesicles isolated from human small intestine indicate that the transfer of D-glucose across the membrane occurs via a Na § dependent transport system. Similar results were reported recently by Turner et al. [28] for studies with brush border membrane vesicles isolated from human renal cortex. The sodium dependent transport system catalyses a cotransport of sodium and glucose, as indicated by the stimulatory effect of an inside negative membrane diffusion potential and by the specific stimulation of sodium on the movement of glucose. This influence of the membrane potential on the flux of a neutral compound can only be explained by a coupling of the non-electrolyte flux to an electrolyte flux, in our case by coupling of glucose and sodium flux. The stimulation of glucose uptake by an inside negative membrane potential demonstrate that either the ternary carrier complex is positively or the free carrier negatively charged and has consequences for an analysis of the driving forces for sodium dependent glucose transport. The driving force exerted by sodium on rheogenic (potential sensitive) transport mechanisms is not the chemical concentration difference but the electrochemical potential
247
difference for sodium across the brush border membrane [17]. An important feature to be recognized in leaky epithelia like small intestine is the high sodium conductance of the "tight" intercellular junctions, as indicated by both electrical and isotope flux measurements [10, 24]. In the absence of sodium in the luminal perfusate, sodium can still leak out from the intercellular spaces into the lumen and serve as a cosubstrate for sodium driven transport processes. Therefore, the absence of sodium in the lumen does not necessarily imply its absence at the transport sites, and as such does not rule out an electrochemical gradient of sodium across the brush border membrane as effective driving force for sodium coupled transport mechanism. Therefore, conflicting results showing either sodium independence [22] or sodium dependence [21] of human intestinal glucose transport in vivo might be due to inadequate experimental techniques. It could be possible that in perfusion experiments small amounts of sodium in the microclimate of the transport sites in conjunction with an electrical potential difference suffice as driving force for glucose transport across the luminal membrane. Thus, in vivo perfusion results will strongly be dependent on the success in manipulating this microclimate, i.e. in overcoming the effect of unstirred layers. After meals nutrients will most probably be present in the intestinal lumen in higher concentrations than in the intestinal epithelial fluid or in the blood. In such a situation, active transport systems for nutrients would not be required, since influx of nutrients will be passive, driven by the concentration difference of the nutrients itself. However, the situation of higher luminal concentrations is transient. Most of the day, the concentration of nutrients (amino acids, sugars) in the interstitial fluid and in the blood will exceed the concentration in the lumen. According to diffusion laws nutrients would now be lost to the lumen. It is in such time periods, that active absorptive transport systems start to play their important role. They will prevent the loss of substrates used for needs of the body. Sodium coupled transport systems are part of active transepithelial transport mechanisms. Via flux coupling of glucose, and sodium, glucose can be transported across the luminal membrane against its concentration gradient driven by the electrochemical potential difference for sodium. Thereby a high intracellular glucose concentration will build up within the epithelial cells and provide the driving force for passive efflux from the cell into the interstitium via a facilitated diffusion type system [12]. The electrochemical potential difference for sodium across the luminal membrane is maintained by the action of the Na+-K § ATPase located in the contraluminal cell side [11,12].
248
Acknowledgements. We are grateful to Professor Dr. K. J. Ullrich and to Professor Dr. R. Kinne for valuable discussion during the preparation of the manuscript. We thank Mrs. I. Rentel and Mrs. U. Silz-Riebandt for the excellent art work of the figures.
Pfliigers Arch. 373 (1978)
15.
16. REFERENCES 1. Berner, W., Kinne, R.: Transport of p-aminohippuric acid by plasma membrane vesicles isolated from rat kidney cortex. Pfliigers Arch. 361, 269-277 (1976) 2. Berner, W., Kinne, R., Murer, H.: Phosphate transport into brush border membrane vesicles isolated from rat small intestine. Biochem. J. 160, 467--474 (1976) 3. Caspary, W. F., Crane, R. K. : Inclusion of L-glucose within the specificity limits of the active sugar transport system of hamster small intestine. Biochim. Biophys. Acta 163, 395-400 (1968) 4. Crane, R . K . : N a + dependent transport in the intestine and other animal tissues. Fed. Proc. 24, 1000-1006 (1965) 5. Crane, R. K.: Absorption of sugars. In: Handbook of Physiology, Section 6, Vol. III, (C. F. Code, ed.), pp. 13231352. Washington D. C. : American Physiological Soc., 1968 6. Dugas, M. C., Ramaswamy, K., Crane, R. K. : An analysis of the D-glucose influx kinetics of in vitro hamster jejunum, based on considerations of the masstransfer coefficient. Biochim. Biophys. Acta 382, 576-589 (1975) 7. Esposito, G., Faelli, A., Capraro, V.: Sugar and electrolyte absorption in rat intestine perfused in vivo. Pfltigers Arch. 340, 335-348 (1973) 8. Evers, J., Murer, H., Kinne, R. : Phenylalanine uptake in isolated renal brush border vesicles. Biochim. Biophys. Acta 426, 5 9 8 615 (1976) 9. F6rster, H., Hoos, I.: The excretion of sodium during the active absorption of glucose from the perfused small intestine of rats. Hoppe-Seyler's Z. Physiol. Chem. 353, 8 8 - 9 4 (1972) 10. Fr6mter, E., Diamond, J.: Route of passive ion permeation throught epithelia. Nature New Biol. 235, 9 - 13 (1972) 11. Fujita, M., Matsui, H., Natano, K., Nakao, M.: Differentiai isolation of microvillus and basolateral membranes from intestinal mucosa. Biochim. Biophys. Acta 274, 336-347 (1972) 12. Hopfer, U., Sigrist-Nelson, K., Murer, H.: Intestinal sugar transport: Studies with isolated plasma membranes. Ann. N.Y. Acad. Sci. 264, 414-427 (1975) 13. Hopfer, U., Nelson, K., Perrotto, J., Isselbacher, K. J. : Glucose transport in isolated brush border membrane from rat small intestine. J. Biol. Chem. 248, 2 5 - 3 2 (1973) 14. Kinne, R., Murer, H., Kinne-Saffran, E., Thees, M., Sachs, G. : Sugar transport by renal plasma membrane vesicles. I.
17.
18.
19.
20. 2l. 22.
23.
24.
25.
26.
27. 28.
Characterization of the systems in the brush border microvilli and the basal-lateral plasma membranes. J. Membrane Biol. 21, 375-395 (1975) Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J.: Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265-275 (1951) Murer, H., Hopfer, U.: Demonstration of electrogenic Na +dependent D-glucose transport in intestinal brush border membranes. Proc. Natl. Acad. Sci. U.S.A. 71,484-488 (1974) Murer, H., Kinne, R.: Sidedness and coupling of transport processes in small intestinal and renal epithelia. In: Biochemistry of membrane transport. Proc. FEBS Symposium, (G.Semenza, E. Carafoli, eds.), pp. 292-304. Berlin-Heidelberg-New York: Springer 1977 Murer, H., Ammann, E., Biber, J., Hopfer, U.: The surface membrane of intestinal epithelial cell. I. Localisation of adenylcyclase. Biochim. Biophys. Acta 433, 509-519 (1976) Murer, H., Hopfer, U., Kinne-Saffran, E., Kinne, R. : Glucose transport in isolated brush border and lateral-basal plasma membrane vesicles from intestinal epithelial cells. Biochim. Biophys. Acta 345, 170- 179 (1974) Neale, R. J., Wiseman, G. : Active intestinal absorption of Lglucose. Nature 218, 473-474 (1968) Olson, W. A., Ingelfinger, F. J. : The role of sodium in intestinal glucose absorption in man. J. Clin. Invest. 47, 1133 - 1142 (1968) Saltzman, D. A., Rector, F. C., Fordtran, J. S.: The role of intraluminal sodium in glucose absorption in vivo. J. Clin. Invest. 51, 876-885 (1972) Schmitz, J., Preiser, H., Maestracci, D., Ghosh, B. K., Cerda, J. J., Crane, R. K. : Purification of the human intestinal brush border membrane. Biochim. Biophys. Acta 323, 98 - 112 (1973) Schultz, S. G., Frizzel, R. A.: An overview of intestinal absorptive and secretory processes. Gastroenterology 63, 161 170 (1972) Sigrist-Nelson, K., Hopfer, U.: A distinct D-fructose transport system in isolated brush border membrane. Biochim. Biophys. Acta 367, 247-254 (1974) Sigrist-Nelson, K., Murer, H., Hopfer, U. : Active alanine transport in isolated brush border membrane. J. Biol. Chem. 250, 5674- 5680 (1975) Stein, W. D.: In: The movement of molecules across cell membranes, p.283. New York-London: Academic Press 1967 Turner, R. J., Silverman, M.: Comparison of sugar uptake kinetics into dog and human renal brush border vesicles. Fed. Proc. 36, 593 (1977)
Received June 22, 1977
Pfl~gers Arch. 373, 243-248 (1978)
EuropeanJournal of Physiology 9 by Springer-Verlag 1978
Sugar Transport by Brush Border Membrane Vesicles Isolated from Human Small Intestine H. LUCKE, W. BERNER*, H. MENGE**, and H. MURER Max-Planck-Institut f/Jr Biophysik, Kennedyallee 70, D-6000 Frankfurt (Main), Federal Republic of Germany
Abstract. Uptake of D- and L-glucose, and fructose by purified brush border membrane vesicles isolated from human small intestine was studied using a rapid filtration technique. The uptake of D-glucose by the vesicles was osmotically sensitive and represented transport into an intravesicular space and not binding to the membranes. Transport of both, D- and L-glucose was inhibited by phlorizin. Uptake of D-fructose in~Lothe brush border vesicles was not stimulated by sodium. In the presence of a sodium gradient D-glucose was taken up 5 times faster than L-glucose. The amount of D-glucose transported into the vesicles in the presence of a sodium gradient was transiently higher than the amount of D-glucose taken up at equilibrium (overshoot). D-Glucose transport was stimulated only by a sodium gradient; other monovalent cations had no effect. In the presence of a sodium gradient D-glucose transport was increased by the lipophilic anion thiocyanate and decreased by the nearly impermeable anion sulfate as compared with uptake of D-glucose in the presence of a sodium chloride gradient. This indicates an influence of the electrical membrane potential on the sodimn coupled non-electrolyte transport. Key words: Man - Intestine - Brush border - Sugar - Transport.
INTRODUCTION In the "sodium gradient hypothesis" Crane postulated that a molecular coupling between sodium and glucose flux in the brush border membrane was responsible for * From the Department of Surgery, Uni'~ersityHospital, D-3000 Hannover, Federal Republic of Germany ** From the Department of Medicine, University Hospital, D_3550 Marburg/Lahn, Federal Republic of Germany
intestinal active sugar transport [4, 5]. However, it was difficult to verify the physiological significance of this mechanism by in vivo perfusion experiments using laboratory animals [7,9] and man [21,22]. The differences in the interpretation of the results obtained by studies with the intact epithelial tissue in vivo can be explained by methodological difficulties in controlling different parameters such as paracellular fluxes and cellular metabolism. In particular, large unstirred layers make it almost impossible to determine substrate and cosubstrate concentrations at the transport sites itself [6]. It is therefore very difficult to get data from in vivo perfusion experiments which allow definition of the driving forces of a Na+-gradient on Na + coupled transport processes. In the last few years methods have been developed to study directly the role of the brush border membrane in reabsorptive processes. Glucose, fructose, amino acid, inorganic phosphate and sodium transport systems have been demonstrated in vesiculated brush border membranes isolated from the small intestine of rats [2, 12, 13,19,25,26]. It has been shown, that the transport of glucose, amino acids and inorganic phosphate is stimulated by sodium via flux coupling of the cosubstrate sodium with the different substrates. Similar experiments were also carried out with brush border membrane preparations isolated from renal proximal tubule [8, 14]. The aim of the present contribution is to explore whether these well documented mechanisms of sodium dependent non-electrolyte movement play also a role in human intestinal glucose absorption. For that we have undertaken the study of glucose transport in brush border membrane vesicles isolated from human small intestine. In such preparations the influence of cellular metabolism is avoided, and since the composition of intra- and extracellular fluid can be easily manipulated, the driving forces for glucose can be defined.
0031-6768/78/0373/0243/$ 01.20
244
The data reported in this contribution show the presence of a cotransport system for sodium and Dglucose (and L-glucose) in the human brush border membrane similar to that described in brush border membranes isolated from laboratory animals.
Pfltigers Arch. 373 (1978) Vibrated epithelial cell suspension
METHODS AND MATERIALS Isolation of Brush Border Membrane Vesicles. 25 cm long pieces of histologically normal human upper jejunum, which were obtained from 2 patients in the course of surgery for tumors involving other tissues of gastrointestinal tract, were rinsed with Ringer solution and kept frozen at minus 40~ C. For membrane isolation 20 g of the frozen material was thawed in Ringer solution at room temperature. The muscularis mucosae was separated from the mucosal layer by dissection. The dissected mucosal pieces were placed in 60 ml of mannitol buffer (300 mM mannitol, 12mM Tris-HC1, pH 7.1) and disrupted in a vibro-blendor (Chemap AG, M/innedorf/ZH, Switzerland). The suspension obtained by vibration of the mucosal pieces was filtered through a nylon sieve (pore size 0.8ram). As indicated by phase contrast microscopy the filtered suspension contained mainly epithelial cells, brush border fragments and free nuclei. This suspension of epithelial cells was homogenized at 0 ~ C in hypotonic medium as described in scheme 1. Membrane isolation was performed by a modification of the procedure of Schmitz et al. [23] as indicated in scheme 11 All the centrifugation steps were carried out in a Sorvall RC 2B refrigerated centrifuge (rotor SS34).
9 discard pellet 1 Supernatant 1
Transport Studies
centrifuge at 20000g for 30min ~discard supernatant 2
Pellet 2
resuspend in 70 ml of mannitol buffer used for transport studies (100 mM mannitol, 20 mM HEPES2-Tris, pH7.4) with a glass teflon homogenizer (10 strokes at 1400rpm); add CaCIz to a final concentration of 10 mM; after 15 min centrifuge at 7500 g for 15 min ,discard pellet 3
Supe}natant 3
centrifuge at 20000 g for 25 min discard supernatant 4
Pellet 4
Protein Concentrations and Enzyme Activities Protein determination was carried out according to Lowry et al. [15] with bovine serum albumin as standard (Behring-Werke, Marburg, W.-Germany). Alkaline phosphatase (EC 3.1.3.1.), an enzyme marker for the intestinal brush border membrane was measured as described by Berner et al. [1]. K+-stimulated pnitrophenyl-phosphatase (EC 3.1.3.41), a marker of the contraluminal plasma membrane, was assayed as reported by Murer et al. [18].
after addition of 240 ml icecold bidest H20 to 60 ml of the vibrated epithelial cell suspension (in a buffer containing 300raM mannitol, 12 mM Tris-HC1, pH 7.1) homogenize with an Universal blendor (MX 32; Braun GmbH,. Melsungen, W.-Germany), 3.5rain at top speed; add CaC12 to a final concentration of 10raM; after 15min centrifuge at 7500g for 15 min
resuspend in 2ml of mannitol buffer (as above) by sucking the suspension 7 times through a stainless steel needle (0.9 x 38 mm) into a plastic syringe; centrifuge at 2600 g for 5 min discard pellet 5
+
Supernatant 5
centrifuge at 15000g for 20min in an Eppendorf table centrifuge (3200) discard supernatant 6
Pellet 6
final brush border vesicle preparation
Scheme 1. Preparation of brush border membrane vesicles from small intestinal epithelial cells
Uptake of labelled compounds by isolated brush border membrane vesicles were measured by a Millipore filtration technique as described previously [13,8]. The exact compositions of the incubation media are given in the legends to the figures.
Materials The labelled compounds were purchased from New England Nuclear Corp. (Boston, MA, U.S.A.). All other chemicals used in the experiments were obtained from Merck (Darmstadt, W.-Germany) with the exceptions of HEPES 2 (Serva, Heidelberg, W.-Germany) and phlorizin (Roth, Karlsruhe, W.-Germany). All chemicals were of the highest purity available. 1 A similar isolation procedure was developed recently for the isolatiotl of brush border membranes from rabbit and rat small intestine (Kessler, M., Acuto, O., Storelli, C., Murer, H., Mfiller, M., Semenza, G. : Biochim. Biophys. Acta, submitted for publication) 2 HEPES: N-2-hydroxyethylpiperazine-N'-ethanesulphonic acid
RESULTS
A. Purity of Brush Border Membrane Vesicles The specific activity of the brush border membrane m a r k e r e n z y m e , a l k a l i n e p h o s p h a t a s e [12,23], w a s 11 t i m e s h i g h e r i n t h e f i n a l m e m b r a n e f r a c t i o n as c o m p a r e d w i t h t h e s t a r t i n g h o m o g e n a t e ( s h o w n i n T a b l e 1). K+-stimulated p-nitrophenyl-phosphatase was only slightly enriched in the purified brush border memb r a n e ( 1 . 6 - f o l d ) , s u g g e s t i n g little c o n t a m i n a t i o n b y b a s a l - l a t e r a l m e m b r a n e s [12].
245
H, Liicke et al. : Human Intestinal Sugar Transport Table 1. Enrichment of marker enzymes in the brush border vesicle preparation MW + S.D. n = 4 Total homogenate (mU/mg of protein)
Vesicle fraction (mU/mg of protein)
Enrichment factor
106.58 + 31.64 4.82 • 5.42
1196.65 +_ 444,83 5.81 +_ 4.69
11.16 _+ 1.79 1.61 • 0.64
Alkaline Phosphatase K+-stimulatedp-nitrophenyl-phosphatase
9 NaSCN gradient
~
I
oNoSCN gradient t D-glucose uptake + 0.SmM phlorizin 9 KSCN gradient § 22NG
e NaSCN gradient
~ 120._ ~_
9 K SCN gradient
~-
uptake
--O--
"6 160 I.,~ 0
c
/
5. E .-~ 120
o
~s
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o9
5
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~, |
~ 2
~o-
80 t
o~
o
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c_
0-
&O -
9
,
0-
0
l
//a__
610
2
Incubation time [min]
120
o Na SCN gradient o NQSCN gradient
-
+0 5 cam phlorizin
~
~g O
--o--
80-
1
2 Incubation time [mini
60
Fig. 2. Time course of D-fructose uptake by brush border membranes. The vesicles were loaded with a buffer containing 100 mM mannitol and 20raM HEPES/Tris (pH7.4) and incubated in a medium containing 0.1mM (3H) fructose and 100raM NaSCN (E]) or 100mM KSCN ( I ) , respectively. Fructose uptake is expressed as percentage of the amount taken up by the vesicles after 60 min of incubation time in the NaSCN (1:5) medium, which amounted to 234 pmoles/mg of protein
~
2o
p~
J
--A--
9 KSCN gradient
121_221
/~
I
0
~0C
/ ~
~
~
#
O0
1 2 fncubcltion time [mi,q]
do
Fig. I. Time course of D-, L-glucose and 2ZNa uptake by human intestinal brush border vesicles. Membrane vesicles isolated according to scheme 1 and loaded with a buffer containing 100mM mannitol and 20 m M HEPES/Tris (pH 7.4) were incubated at 25 ~ C in a medium containing 0.1 m M D-(Ir (a) or 0.1raM L(3H)glucose (b), 100 m M mannitol, 20ram HEPES/Tris (pH 7.4), 100ram NaSCN (O) or 100raM Z2NaSCN ( + ) or i 0 0 m M NaSCN plus 0.5 mM pMorizin (O) or 100 m M KSCN ( , ) . Glucose uptake is expressed as percentage o f the amount taken up by the membranes after 60 min of incubation in the NaSCN (O) medium. The absolute value for the NaSCN (e) medium was 241 pmoles/mg of protein (a) and 204pmoles/mg of protein (b) after 60 min of incubation, for 22NaSCN ( + ) it was II7nmoles/mg of protein after 60min of incubation
B. Transport Properties When the two stereoisomers D- and L-glucose were added to the isolated brush border vesicles, different time courses of uptake were obtained (Fig. 1a, b). The amount of D-glucose taken up by the vesicles in the presence of a sodium gradient during the first 60 s of
incubation was about 4.6 times higher than that of eglucose. This difference was reduced by about half if sodium was replaced by potassium. In the presence of a NaSCN gradient D-glucose uptake showed an "overshoot" phenomenon, during which the intravesicular glucose concentration transiently exceeded the concentration of glucose in the incubation medium. The "overshoot" arose because of the persistance of a sodium gradient when the intravesicular glucose had already reached the concentration of the incubation medium. D-Glucose concentration within the vesicles reached the concentration of the medium in less than 10 s, whereas sodium reached only 30 ~ of equilibrium after this incubation period (Fig. 1a). The initial uptake olDglucose was stimulated 3.6 times in the presence of a sodium gradient compared with the presence of a potassium gradient. L-Glucose uptake was only slightly stimulated by sodium (1.5-fold). D-Fructose, which is thought from experiments with animal preparations to be transported by sodium-independent "facilitated diffusion" [25], showed also no sodium stimulated transport in human intestinal brush border vesicles (Fig. 2).
246
Pfltigers Arch. 373 (1978)
//
~_ 80-
60-
o D-gLucose T z; D-glucose plus O5 mM ph[orizin -t~ 9 D-gtucose(rninus O-glucose with 9hlorizin) i
-.~
t
~6
./ /
/.0i
20/ /
/ t
/
-~cL 2000
9
o
t
/ / ~-
1000
& 0 0
i
i
i
i
I
I
i
1
2
3
4
5
6
7
Osmo[flrity
0
-1
1
2 3 D- glucose rmM]
Z~
5
Fig. 3. Influence of medium osmolarity on D-glucose uptake by isolated brush border membrane vesicles. The uptake of 0.1 mM D(l~C)glucose was determined in the presence of I00 mM mannito[, 20 mM HEPES/Tris (pH 7.4), t0 mM NaCI and sufficientcetlobiose to give the indicated osmolarity. The values given represent equilibrium values obtained after 15min of incubation
Fig.4. Influenceof D-glucoseconcentration on D-glucoseuptake by brush border membrane vesicles. The incubation medium contained 100mM mannitol, 20 mM HEPES/Tris (pH 7.4), 100mM NaSCN and D-glucose (O), or D-glucose plus 0.5 mM phlorizin (A) at the indicated concentrations(Fig.4a). The specificsodium dependent Dglucose uptake ([3) is obtained by the total uptake (O) minus the phlorizin inhibited part (A). The amounttaken up by the vesiclesafter 60 s is given
The amount of D-glucose taken up by the vesicles at equilibrium was in inverse proportion to the osmolarity of the incubation medium (Fig.3). These findings indicate the presence of osmotically reactive membrane vesicles and secondly transport of glucose into an intravesicular space. Such findings cannot be explained by binding of substrates to or incorporation of substrates into the membrane. The amount of o- and L-glucose taken up after 60 s into brush border membrane vesicles increased in curvilinear fashion when the glucose concentration in the incubation medium was increased (Fig. 4). In the presence of phlorizin, which inhibits the sodium dependent glucose transport [5,27], a smaller but linear Dglucose uptake was observed which might indicate uptake by simple diffusion. The carrier mediated sodium dependent o-glucose transport is supposed to be the difference of the total uptake minus the phlorizin inhibited part of o-glucose uptake. In the concentration range used in this experiment (Fig.4) no complete saturation of o-glucose was observed. This indicates that the apparent Michaelis constant of the transport system (Kin) must be rather high (above 5 mM). This low affinity of the transport system for glucose is further supported by kinetic analysis (data not shown) of the initial (15 s) uptake rates (Lticke, H., Berner, W., Menge, H., Murer, H. : unpublished results). L-Glucose uptake was also inhibited by phlorizin as it was slightly stimulated by sodium (Fig. 1 b). From
Table 2. Effect of cations on D-glucoseuptake into intestinal brush border membrane vesicles (amount taken up during the first 60s). The experiments were carried out in an incubation mediumcontaining 100raM mannitol, 20mM HEPES/Tris (pH7.4), 0.1 mM D(14C)glucose and different salt gradients as given in the table Salt in the incubation medium
D-Glucose uptake
(0.1 M)
(pmoles/mg of protein)
Choline chloride LiC1 NaC1 KCI RbC1 CsCI
43.5 43.7 114.5 54.4 41.1 40.9
these experiments the existence of carrier mediated Lglucose uptake could be inferred [3,12,20]. Table 2 shows the effect of various cations on oglucose transport by brush border membrane vesicles. The highest stimulation was seen with sodium. Among the other cations tested no stimulatory effect could be detected as compared with the uptake in the presence of a choline gradient. As shown in Table 3 replacement of CI- in the presence of a sodium gradient by a more permeant anion such as S C N - stimulated the initial uptake of glucose. As we have demonstrated earlier in experiments with rat small intestinal preparations this
H. Liicke et al. : Human Intestinal Sugar Transport Table3. Effect of anion replacement on D-glucose uptake into intestinal brush border membrane vesicles (amount taken up during the first 60 s). The incubation medium contained 100 mM mannitol, 20 mM HEPES/Tris (pH 7.4), 0.1 mM D-(l~C)glucose and different salt gradients as indicated in the table Salt in the incubation medium
D-Glucose uptake (pmoles/mg of protein)
0.1 M NaSCN 0.1M NaC1 0.05 M Na2SO 4 + 0.05 M mannitol
224.7 114.5 94.4
effect is best explained by the assumption of a hyperpolarisation (vesicle inside negative) of the membrane potential in the presence of a SCN- gradient [8, 16]. If the membrane potential is reversed by the use of SO l instead of C1-, in the presence of a sodium gradient the rate of uptake is markedly reduced. These results strongly point to an involvement of the membrane potential in the sodium dependent transmembranal movement of glucose and to a rheogenicity of the flux coupling mechanism between sodium and glucose.
DISCUSSION Our studies on the D-glucose transport by brush border vesicles isolated from human small intestine indicate that the transfer of D-glucose across the membrane occurs via a Na § dependent transport system. Similar results were reported recently by Turner et al. [28] for studies with brush border membrane vesicles isolated from human renal cortex. The sodium dependent transport system catalyses a cotransport of sodium and glucose, as indicated by the stimulatory effect of an inside negative membrane diffusion potential and by the specific stimulation of sodium on the movement of glucose. This influence of the membrane potential on the flux of a neutral compound can only be explained by a coupling of the non-electrolyte flux to an electrolyte flux, in our case by coupling of glucose and sodium flux. The stimulation of glucose uptake by an inside negative membrane potential demonstrate that either the ternary carrier complex is positively or the free carrier negatively charged and has consequences for an analysis of the driving forces for sodium dependent glucose transport. The driving force exerted by sodium on rheogenic (potential sensitive) transport mechanisms is not the chemical concentration difference but the electrochemical potential
247
difference for sodium across the brush border membrane [17]. An important feature to be recognized in leaky epithelia like small intestine is the high sodium conductance of the "tight" intercellular junctions, as indicated by both electrical and isotope flux measurements [10, 24]. In the absence of sodium in the luminal perfusate, sodium can still leak out from the intercellular spaces into the lumen and serve as a cosubstrate for sodium driven transport processes. Therefore, the absence of sodium in the lumen does not necessarily imply its absence at the transport sites, and as such does not rule out an electrochemical gradient of sodium across the brush border membrane as effective driving force for sodium coupled transport mechanism. Therefore, conflicting results showing either sodium independence [22] or sodium dependence [21] of human intestinal glucose transport in vivo might be due to inadequate experimental techniques. It could be possible that in perfusion experiments small amounts of sodium in the microclimate of the transport sites in conjunction with an electrical potential difference suffice as driving force for glucose transport across the luminal membrane. Thus, in vivo perfusion results will strongly be dependent on the success in manipulating this microclimate, i.e. in overcoming the effect of unstirred layers. After meals nutrients will most probably be present in the intestinal lumen in higher concentrations than in the intestinal epithelial fluid or in the blood. In such a situation, active transport systems for nutrients would not be required, since influx of nutrients will be passive, driven by the concentration difference of the nutrients itself. However, the situation of higher luminal concentrations is transient. Most of the day, the concentration of nutrients (amino acids, sugars) in the interstitial fluid and in the blood will exceed the concentration in the lumen. According to diffusion laws nutrients would now be lost to the lumen. It is in such time periods, that active absorptive transport systems start to play their important role. They will prevent the loss of substrates used for needs of the body. Sodium coupled transport systems are part of active transepithelial transport mechanisms. Via flux coupling of glucose, and sodium, glucose can be transported across the luminal membrane against its concentration gradient driven by the electrochemical potential difference for sodium. Thereby a high intracellular glucose concentration will build up within the epithelial cells and provide the driving force for passive efflux from the cell into the interstitium via a facilitated diffusion type system [12]. The electrochemical potential difference for sodium across the luminal membrane is maintained by the action of the Na+-K § ATPase located in the contraluminal cell side [11,12].
248
Acknowledgements. We are grateful to Professor Dr. K. J. Ullrich and to Professor Dr. R. Kinne for valuable discussion during the preparation of the manuscript. We thank Mrs. I. Rentel and Mrs. U. Silz-Riebandt for the excellent art work of the figures.
Pfliigers Arch. 373 (1978)
15.
16. REFERENCES 1. Berner, W., Kinne, R.: Transport of p-aminohippuric acid by plasma membrane vesicles isolated from rat kidney cortex. Pfliigers Arch. 361, 269-277 (1976) 2. Berner, W., Kinne, R., Murer, H.: Phosphate transport into brush border membrane vesicles isolated from rat small intestine. Biochem. J. 160, 467--474 (1976) 3. Caspary, W. F., Crane, R. K. : Inclusion of L-glucose within the specificity limits of the active sugar transport system of hamster small intestine. Biochim. Biophys. Acta 163, 395-400 (1968) 4. Crane, R . K . : N a + dependent transport in the intestine and other animal tissues. Fed. Proc. 24, 1000-1006 (1965) 5. Crane, R. K.: Absorption of sugars. In: Handbook of Physiology, Section 6, Vol. III, (C. F. Code, ed.), pp. 13231352. Washington D. C. : American Physiological Soc., 1968 6. Dugas, M. C., Ramaswamy, K., Crane, R. K. : An analysis of the D-glucose influx kinetics of in vitro hamster jejunum, based on considerations of the masstransfer coefficient. Biochim. Biophys. Acta 382, 576-589 (1975) 7. Esposito, G., Faelli, A., Capraro, V.: Sugar and electrolyte absorption in rat intestine perfused in vivo. Pfltigers Arch. 340, 335-348 (1973) 8. Evers, J., Murer, H., Kinne, R. : Phenylalanine uptake in isolated renal brush border vesicles. Biochim. Biophys. Acta 426, 5 9 8 615 (1976) 9. F6rster, H., Hoos, I.: The excretion of sodium during the active absorption of glucose from the perfused small intestine of rats. Hoppe-Seyler's Z. Physiol. Chem. 353, 8 8 - 9 4 (1972) 10. Fr6mter, E., Diamond, J.: Route of passive ion permeation throught epithelia. Nature New Biol. 235, 9 - 13 (1972) 11. Fujita, M., Matsui, H., Natano, K., Nakao, M.: Differentiai isolation of microvillus and basolateral membranes from intestinal mucosa. Biochim. Biophys. Acta 274, 336-347 (1972) 12. Hopfer, U., Sigrist-Nelson, K., Murer, H.: Intestinal sugar transport: Studies with isolated plasma membranes. Ann. N.Y. Acad. Sci. 264, 414-427 (1975) 13. Hopfer, U., Nelson, K., Perrotto, J., Isselbacher, K. J. : Glucose transport in isolated brush border membrane from rat small intestine. J. Biol. Chem. 248, 2 5 - 3 2 (1973) 14. Kinne, R., Murer, H., Kinne-Saffran, E., Thees, M., Sachs, G. : Sugar transport by renal plasma membrane vesicles. I.
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Characterization of the systems in the brush border microvilli and the basal-lateral plasma membranes. J. Membrane Biol. 21, 375-395 (1975) Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J.: Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265-275 (1951) Murer, H., Hopfer, U.: Demonstration of electrogenic Na +dependent D-glucose transport in intestinal brush border membranes. Proc. Natl. Acad. Sci. U.S.A. 71,484-488 (1974) Murer, H., Kinne, R.: Sidedness and coupling of transport processes in small intestinal and renal epithelia. In: Biochemistry of membrane transport. Proc. FEBS Symposium, (G.Semenza, E. Carafoli, eds.), pp. 292-304. Berlin-Heidelberg-New York: Springer 1977 Murer, H., Ammann, E., Biber, J., Hopfer, U.: The surface membrane of intestinal epithelial cell. I. Localisation of adenylcyclase. Biochim. Biophys. Acta 433, 509-519 (1976) Murer, H., Hopfer, U., Kinne-Saffran, E., Kinne, R. : Glucose transport in isolated brush border and lateral-basal plasma membrane vesicles from intestinal epithelial cells. Biochim. Biophys. Acta 345, 170- 179 (1974) Neale, R. J., Wiseman, G. : Active intestinal absorption of Lglucose. Nature 218, 473-474 (1968) Olson, W. A., Ingelfinger, F. J. : The role of sodium in intestinal glucose absorption in man. J. Clin. Invest. 47, 1133 - 1142 (1968) Saltzman, D. A., Rector, F. C., Fordtran, J. S.: The role of intraluminal sodium in glucose absorption in vivo. J. Clin. Invest. 51, 876-885 (1972) Schmitz, J., Preiser, H., Maestracci, D., Ghosh, B. K., Cerda, J. J., Crane, R. K. : Purification of the human intestinal brush border membrane. Biochim. Biophys. Acta 323, 98 - 112 (1973) Schultz, S. G., Frizzel, R. A.: An overview of intestinal absorptive and secretory processes. Gastroenterology 63, 161 170 (1972) Sigrist-Nelson, K., Hopfer, U.: A distinct D-fructose transport system in isolated brush border membrane. Biochim. Biophys. Acta 367, 247-254 (1974) Sigrist-Nelson, K., Murer, H., Hopfer, U. : Active alanine transport in isolated brush border membrane. J. Biol. Chem. 250, 5674- 5680 (1975) Stein, W. D.: In: The movement of molecules across cell membranes, p.283. New York-London: Academic Press 1967 Turner, R. J., Silverman, M.: Comparison of sugar uptake kinetics into dog and human renal brush border vesicles. Fed. Proc. 36, 593 (1977)
Received June 22, 1977
