INTESTINAL SUGAR TRANSPORT: STUDIES WITH ISOLATED PLASMA MEMBRANES* U. Hopfer Department of Anatomy School of Medicine, and Developmental Biology Center Case Western Reserve University Cleveland, Ohio 441 06

K. Sigrist-Nelson Laboratory f o r Biochemistry Federal Znstitute of Technology Zurich, Switzerland

H. Murer Max Planck Institute f o r Biophysics Frankfurt, Federal Republic of Germany

INTRODUCTION The absorptive epithelium of the small intestine is specialized in the transfer of nutrients from the lumen to the blood and lymph vessels. Traditionally, transport studies have been carried out in intestinal preparations with intact epithelial cells and precautions were taken to keep cells viable during the time of measurements. The information obtained in this way led to concepts about the nature of transport processes, specificity of transport systems, input of energy, etc., and there are several recent review articles on these Unfortunately, because of the complexity of eukaryotic cells, ambiguities often exist in the interpretation of transport data, and this has resulted in considerable controversies, especially with respect to the nature of the energy supply for uphill Dglucose transport and the role of Na' in this process.'-' Is chemical energy, in the form of ATP for example, directly involved in Dglucose uptake by epithelial cells or is the energy to drive sugar transport provided indirectly via cation gradients? Can useful energy be gained for the concentrative uptake of nutrients from ion gradients? In order to define the mechanisms in sugar transport more clearly we have gone to a subcellular system of isolated plasma membranes. Deliberate efforts were made to break up the intestinal epithelial cell and purify separately its two types, Le., the luminal brush border (microvillous) and the contraluminal lateral-basal plasma membrane. This approach, by necessity, involves disruption of the surface membrane and thereby destruction of its barrier property. Since any assay for transport depends on the latter property of membranes, it became necessary to find conditions under which the membrane fragments *This research was supported by Grants No. 3.511.71 and 3.0090.73 from the Swiss Nationalfonds. 414

Hopfer et al.:

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Intestinal Sugar Transport

would form vesicles and reseal after they had been isolated from the cell. This paper will discuss some of the considerations that enabled us to carry out the studies on intestinal sugar transport in isolated plasma membranes as well as the conclusions derived from them. Many of the experimental details and results have been published.'-"

ISOLATION OF PLASMAMEMBRANES In intestinal epithelial cells the luminal and contraluminal region of the surface membrane have distinctly different chemical and enzymatic composition as well as appearance in the electron m i c ~ - o s c o p e .Therefore, ~~-~~ is was desirable not only to separate plasma membrane from intracellular membranes and organelles, but also to brush borders from lateral-basal membrane. Several methods are available for the isolation of intestinal plasma membranes.', 16-zz FIGURE 1 illustrates the methods that we found convenient and effective in yielding highly purified plasma membrane fractions from rat small intestine. Briefly, for isolation of microvillous membranes intact brush borders are sheared off epithelial cells and isolated by low-speed centrifugation. Subsequently, most of the microfilaments are removed whereby smaller membrane fragments are formed. These fragments are collected by centrifugation at 7.2 x lo5x g min. The purification of plasma membrane enzymes is given in TABLE1. The specific activities of typical brush border enzymes like sucrase (E.C. 3.2.1.48), leucine amino peptidase (E.C. 3.4.1 1.1) , and alkaline phosphate (E.C. 3.1.3.1) are 36-41 times higher in the final membrane as compared with the starting homogenate of intestinal scrapings. If isolated cells are taken a3 basis for comparison the purification of brush border enzymes is about 20-fold. This difference is caused by the presence of other cell types besides enterocytes in the scrapings. Typical enzymes of the lateral-basal plasma mem-

FIGURE I. Preparation of luminal and contraluminal plasma membrane.

0 Na',Kn'- I T P a s c adcnyl cychsc S'nuclcotidase CONTRA-LUMIWIL PM

0 sucrase alh. Pasc k u m i n o peptidare

L U M I N I L PM

416

Annals New York Academy of Sciences TABLE1

ENZYMATIC COMPOSITION

Sucrase* Leucine amino peptidase* Alkaline phosphatase* K +-stimulated phosphatase* Adenyl cyclaset

OF

BRUSHBORDERMEMBRANE

Intestinal Scrapings

Brush Border Membrane

Purification

0.09 0.16 0.08 0.054 36

3.33 5.70 3.18 0.030 32

37 36 41 0.6 0.9

NOTE: All enzyme activities were measured at 37 “C. Sucrase, leucine-aminopeptidase, and Na+, K+-ATPase were determined according to References 7. 58, and 17, respectively. K+stimulated phosphatase was assayed in the presence of 10 mM MgCl,, 5 mM EDTA, and 6 mM p-nitrophenyl phosphate (di-Tris salt) at pH 7.8; the K+-stimulation was calculated from the difference in phosphate release with either 90 mM KCl or 90 mM NaCI. S’-nucleotidase was measured in a medium containing 10 mM MgCl,, and either 5 mM 5’-AMP or 5 mM 3‘-AMP at pH 7.6; the difference in phosphate release from the two nucleotides was contributed to specific 5’-nucleotidase activity. Alkaline phosphatase was assayed in the presence of 2.5 mM MgCl,, 2.5 mM ZnSO, and 5.5 mM p-nitrophenyl phosphate at pH 10.5. Adenyl cyclase was determined in the presence of 4 mM MgCl,, 0.1 mM EDTA, 3 mM ATP, 10 mM theophylline, 10 mM creatine phosphate, 0.1 mg/ml creatine kinase, and 10 mM NaF at pH 7.4. * Activity in pmoles substrate hydrolyzed per min per mg protein. t Activity in pmoles product synthesized per min per mg protein.

brane, e.g., K-stimulated phosphatase (E.C. 3.1.3.) and F--activated adenyl cyclase (E.C. 4.6.1.1.) , are not enriched in the purified brush border membrane (TABLE1) . Their low levels indicate little crosscontamination from the lateralbasal plasma membrane. The physical properties of membrane fragments from the contraluminal region resemble those of smooth intracellular membranes and may be similar to the plasma membranes of other cell types. Therefore, preparation of this membrane starts out with collection of intestinal epithelial cells. The isolated cells are then homogenized and a “crude” plasma membrane fraction is prepared by differential centrifugation. Most of the plasma membranes settle slightly slower than the mitochondria, as described previously.g Further purification of the surface membranes is achieved by flotation of the membranes through a discontinuous gradient of 20-50% sucrose at 18 x loo x g min. Purified lateral-basal plasma membranes are collected at the 20/30% interface 2 shows enzyme and brush border membrane at the 40/50% interface. TABLE activities of the lighter membrane faction (20/ 30% sucrose). Na+,K+-ATPase (E.C. 3.6.1.3), K+-stimulated phosphatase, S’nucleotidase (E.C. 3.1.3.5.), and F--activated adenyl cyclase are enriched 10-14 times over the cell homogenate. All these enzymes are considered to be plasma membrane marker^.'^ Very little activity of brush border enzymes is associated with this fraction, as 2. Hence most of the indicated by sucrase or alkaline phosphatase in TABLE membrane has to be derived from the lateral-basal region. Stirling” actually showed by autoradiography that ouabain, a specific inhibitor of Na’, K-ATPase, binds specifically to this part of the epithelial cell. A membrane fraction enriched in brush border enzymes can be collected at the 40/50% interface from the same gradient (data not shown).

Hopfer et al.: Intestinal Sugar Transport

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TABLE2 ENZYMATIC COMPOSITION OF LATERAL-BASAL PLASMA MEMBRANE

Isolated Cells Na+, K+-ATPase*

0.067 0.017 0.080

K+-stimulated phosphatase* 5'-nucleotidase* Adenyl cyclaset Sucrase* Alkaline phosphatase* ~~~~~

~

Lateral-Basal Plasma Membrane

Purification

0.69 0.23 0.93

10 14

41 0.16

454 0.18

11

0.23

0.12

12

1.1 0.5

~

NOTE: For determination of enzyme activities see TABLE 1. * Activity in pmoles substrate hydrolyzed per min per mg protein. t Activity in pmoles product synthesized per min per mg protein.

TRANSPORT STUDIES

IN ISOLATED

MEMBRANES

Any assay for transport of solutes is contingent upon membranes enclosing a topographical closed space and effectively separating two aqueous compartments. Fortunately, many plasma membranes form vesicles during homogenization. Depending on experimental conditions, the isolated intestinal membranes contain a space from about 0.5 to 10 PI per mg membrane protein. The magnitude of the space has been estimated from the uptake of permeant solutes equilibrating between the suspension medium and the intravesicular space. Size of membrane fragments appears to be one of the factors determining the space. It has been noted that lateral-basal membranes form larger vesicles than the brush border membranes as judged by electronmicroscopy of negatively stained preparations, and this correlates with a greater equilibrium uptake of permeant solutes in lateral-basal membranes (unpublished observation). Osmotic forces also influence the size of the intravesicular compartment. To prevent collapse of this space during an experiment, it is necessary to trap an impermeant solute during formation of the membrane vesicles. We have employed extensively either metabolically inert polyols, like mannitol and sorbitol, or carbohydrates like cellobiose. The osmotic behavior of membrane vesicles can be followed by measuring the equilibrium uptake of permeant solutes such as D-glucose. At equilibrium the intravesicular concentration equals the medium concentration. Hence, the amount of permeant solute per unit weight of the membrane is proportional to the volume when only this parameter is varied. FIGURE 2 shows the equilibrium uptake of labeled D-glucose by different aliquots of the same membrane preparation suspended in media with different osmolarity. The change in the medium osmolarity is achieved by increasing concentrations of the impermeant solute cellobiose. FIGURE 2 demonstrates that D-glucose uptake is indirectly proportional to the medium osmolarity with no uptake on extrapolation to infinity (zero on the inverse osmolarity scale). Thus, the membrane vesicles behave as predicted from osmotic considerations. The equilibrium uptake of other permeant sugars (D-fructose, L-glucose) or neutral amino acids (D- and L-alanine, L-valine) shows identical behavior. However, we have observed that under certain experimental conditions binding of labeled solutes to the membranes takes place in addition to transport into the membrane space."

Annals New York Academy of Sciences

418

FIGURE2. Effect of medium osmolarity on D-glucose uptake by brush border membranes. Preparation of membranes (in 100 mM cellobiose, 1 mM HEPES adjusted with Tris to pH 7.5, and 0.1 mM MgSO,) has been described.' Uptake of &glucose was measured in aliquots of the same membrane preparation from media with the following final composition: NaSCN (25 mM), Tris-HEPES (1 mM), MgSO. (0.1 mM), D-(l-'H)glucose (1 mM) and enough cellobiose to give the indicated osmolarity (shown as inverse osmolarity). Length of incubation: 10 minutes; temperature: 25°C. The line was calculated by regression analysis (correlation coefficient 0.99).

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SUGAR TRANSPORT IN BRUSHBORDERMEMBRANE The existence of transport systems or carriers in biological membranes is usually inferred from such observations as differential rates of transport for structurally similar compounds, saturation of transport rates with increasing substrate concentration, uptake of solutes against a cohcentration gradient, and enhancement of solute flow across the membrane by the flow of other solutes in the same direction (cotransport) or related compounds in the other direction (countertransport). All of these phenomena have been shown to occur with monosaccharides in the intestinal membrane vesicles, testifying to the existence of intact sugar carriers after membrane isolation.', lo FIGURE 3 shows the uptake of D-glucose, D-fructose, and L-glucose by brush border membranes. The sugar transport is followed using labeled monosaccharides and quenching transport with ice-cold buffer (for details see reference 10). Aliquots of membranes, containing the transported sugars, are collected on membrane filters and the retained sugars are determined with a scintillation counter. Other radioactive compounds can be added to the quench solution to get an estimate of the contamination from the incubation medium. Retention of medium sugars is usually negligible. One of the advantages of working with isolated membranes, namely, absence of sugar metabolism, becomes evident at this point. Under our conditions we have never seen any evidence for conversion of the employed labeled sugars to other compounds, and thus radioactivity on the filter is proportional to the amount of the compound originally containing the label. It is obvious from FIGURE 3 that all three monosaccharides are taken up to the same extent on prolonged incubation. This finding is expected for solutes equilibrating into the same membrane space and in the absence of metabolic energy. More interestingly, the transport rates differ considerably for the various monosaccharides and have the following order: Dglucose D fructose L-glucose. It is difficult to draw any conclusions about possible factors determining L-glucose permeability of the membrane since it exhibits the slowest rate. However, specific agencies (carriers) must exist in the membrane that catalyze the higher rates for other sugars in comparison to L-glucose. Inhibition and countertransport experiments with brush border membranes have established that separate carriers exist for D-fructose and for D-glucose

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Hopfer et al.: Intestinal Sugar Transport

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FIGURE 3. Time course of D-glucose (e),D-fructose (m), and L-glucose (0) uptake by brush border membranes. Membranes were prepared in 100 mM D-mannitol, 1 mM HEPES adjusted with Tris to pH 7.5, and 0.1 mM MgSOI. Aliquots of the membrane were incubated in media containing (final concentration) : 100 mM D-mannitol, 1 mM Tris-HEPES, pH 7.5, 0.1 mM MgS04, 100 mM NaSCN and 1 m M of either D-( I-"H)glucose, D-(I-"H)fructose, or L-( I-"C)glucose. Temperature: 25°C. For further details see Reference 10. (From Sigrist-Nelson and Hopfer." By permission of Biochimicn et Biophysica A m . )

and glucalogs including D-galactose.', discussed separately.

The properties of the two carriers are

D-Glucose Transport Intestinal glucose transport differs from that of most other tissues in that is active in the sense of absorption it is dependent on the presence of Na+,25-2T against a concentration gradient,29-'" and is quite specifically inhibited by the glucoside phlorizin."'. 32 Interestingly, all these phenomena have been demonstrated in the isolated brush border membrane,'. li and these similarities have helped to ascertain that the glucose carrier is retained intact through the preparative procedure. Conditions under which D-glucose is transported against a concentration gradient have contributed considerably to our understanding of the mechanism of active nutrient transport in animal cells. Examples of such experiments are given in the upper curves of FIGURES 4 and 5 where the overshooting uptake of D-glucose upon addition of NaSCN represents transient concentrative uptake. In order to understand the forces responsible for the D-glucose rnovement into the vesicles it is necessary to describe the experiments in some detail: The brush border membranes are preincubated with labeled D-glucose ( 1 mM) in the absence of Na'. The sugar equilibrates, although slower than in the presence of Na', between the medium and the intravesicular space as evidenced by constant D-glucose levels after about 10 minutes of the preincubation period. On addition of NaSCN (final concentration 0.1 M ) further uptake of D-glucose takes place before a new, lower equilibrium value is

420

Annals New York Academy of Sciences FIGURE4. Effect of monactin on active D-glUCOSe uptake by brush border membrane. D-GlUCOSe uptake was measured in the absence ( 0 ) or presence of 12.5 pg/ml monactin (0). Membranes were preincubated at 25°C for 12 minutes in the following medium: 100 mM D-mannitol, 0.1 mM MgSO,, 1 mM Tris-HEPES, pH 7.5, 1% ethanol, and 1 mM D-( I-’H)glucose. The arrow indicates the addition of a 1-M solution of NaSCN (final concentration: 0.1 M ) . Uptake measurements were made as usual.3

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reached. The overshooting part is dependent on the establishment of a NaSCN gradient.’ The interpretation of the uptake data is facilitated by the absence of D-glucose metabolism and osmotic behavior of membrane vesicles. In the experi4 and 5 the amount of Pglucose in the membranes increased ments of FIGURE over the equilibrium level after addition of a hypertonic 1 M NaSCN solution, i.e., in the absence of an increase in vesicle volume. The overshoot must therefore represent a higher intravesicular D-glucose concentration as compared to equilibrium, and therewith to medium, since the two are identical for permeant nonelectrolytes. An estimate of the D-glucose gradient (intravesicular/medium concentration) can be obtained from the ratio of the overshooting to the equilibrium uptake. Under optimal conditions, in the presence of a proton gradient instead of a NaSCN gradient, transient accumulation of D-glucose up to ten times the medium concentration has been observed.* Although the concentration gradients are small in comparison to those established by bacterial membranes they are in line with usual findings for intact animal tissue.” Energy for this active transport must have been derived from the salt gradient present at time zero of the incubation period. It is a limited amount and dissipated within about 2 minutes as measured by the equilibration of labeled NaSCN between medium and membranes.”” The time curve of active D-glucose transport is consistent with the limited supply of energy. The relevant findings with respect to the transport mechanism of D-glucose can be summarized as follows’~R : (i) Active D-glucose transport is observed FIGURE 5. Effect of gramicidin D on active D-glucose uptake by brush

border membrane. D-Glucose uptake was measured in the absence ( 0 )or presence of 18 rg/ml gramicidin D (0). Membranes were treated as explained in the legend to FIGURE4. The arrow indicates the addition of a 1-M solution of NaSCN (final con-

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INtestinal sugar transport: studies with isolated plasma membranes.

The luminal and the contraluminal plasma membranes of small intestinal epithelial cells were isolated separately and in the form of membrane vesciles...
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