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Received 27 April 1998; accepted after revision 15 September 1998.
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ABSTRACT |
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-methyl-D-glucoside while Vmax values were significantly reduced in the ileum and in the rectum. All Scatchard plots of specific [3H]phlorizin binding give a straight line, consistent with a single population of binding sites. Phlorizin binding vs. -methyl-D-glucoside maximal transport rates showed a linear correlation.
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INTRODUCTION |
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In the chicken, responses to Na+ loading and depletion consist of homeostatic adjustments that involve the kidney and the intestine (Grubb & Bentley, 1987; Skadhauge, 1993). In the large intestine, low-NaCl maintenance enhances Na+ absorption in the caecal bulb 2-fold (Thomas & Skadhauge, 1989) while in the coprodeum the increase in absorption is 100-fold, the largest observed in vertebrate epithelia as pointed out by Skadhauge (1993). In the rectum - the segment between the ileo-caecal junction and the coprodeum - adaptation to low-NaCl intake decreases ionic paracellular permeability (Amat et al. 1988), opens Na+ channels in the apical membrane (Skadhauge, 1993; Goldstein et al. 1997), increases Na+ uptake across the Na+-H+ exchanger by inducing apical NHE2 expression (Donowitz et al. 1998), reduces K+ secretion (Munck & Munck, 1990) and also modulates Cl- secretion (Clauss et al. 1991).
The intestinal transport of non-electrolytes is also affected by the dietary intake of NaCl. A Na+-dependent phlorizin-sensitive SGLT1-type transporter is present in the brush-border of the small intestine as well as in regions anatomically belonging to the large intestine (Amat et al. 1996; Bindslev et al. 1997; Garriga et al. 1998). In the basolateral membrane there is a Na+-independent, phloretin and cytochalasin B-sensitive GLUT2-type transporter, that can transport D-glucose and D-fructose with relatively low affinity and high capacity (Kimmich & Randles, 1975; Garriga et al. 1997). Combined apical and basolateral transport systems enable enterocytes to reach a 13-fold accumulation ratio (control/phlorizin) for the glucose analogue 3-oxy-methyl-D-glucose (Glu3Me) in the jejunum and more than 20-fold in the ileum, the proximal caecum and the rectum (Ferrer et al. 1994). However, when birds are fed a low-NaCl (LS) diet, the cumulative capacity of the jejunum is reduced in part (Jaso et al. 1995) while in the rectum it is completely abolished (Lind et al. 1980; Árnason & Skadhauge, 1991; Jaso et al. 1995). Since LS diet adaptation induces secondary hyperaldosteronism, most of the effects previously described are believed to be regulated or controlled by aldosterone (Skadhauge, 1993). The direct involvement of other hormones such as arginine-vasotocin or prolactin - suggested to maintain the high hexose/amino acid-Na+ cotransport in the rectum - has been rejected (Árnason & Skadhauge, 1991).
Part of this work was presented to The Physiological Society (Guy's London Meeting) and has been published in abstract form (Moretó et al. 1998).
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METHODS |
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Animals
Male White Leghorn chickens (Gallus gallus domesticus L.) were obtained from a commercial farm (Gibert, Tarragona, Spain) on the day of hatching and maintained in standardized temperature and humidity conditions, with a 18 : 6 h light-dark cycle. The birds had free access to water and a commercial diet (Gallina Blanca Purina, Barcelona, Spain). When chickens were 10 weeks old, they were kept for 14 days on a diet of wheat and barley (1 : 1) containing (in g kg-1): crude protein, 107·9; lipid, 20·5; carbohydrate, 626·4; crude fibre, 100·5; Na+, 0·013; K+, 0·413; and Cl-, 0·346. The metabolizable energy content was 13·2 MJ kg-1. Drinking water contained added NaCl to yield a final concentration of either 200 mM (high-Na+ or HS diet) or 0·015 mM (low-Na+ or LS diet). Manipulation and experimental procedures are in accordance with the Spanish regulations for the use and handling of experimental animals.
Experiments were carried out at the age of 12 weeks. Animals were killed in the morning, without previous withholding of food, by cervical dislocation followed by exsanguination. The intestinal segments were removed, immediately flushed with ice-cold saline containing 0·2 mM phenylmethanesulphonyl fluoride (PMSF), 0·41 µM LiN3 and 0·1 mM benzamidine, and opened lengthwise. The mucosa was scraped, frozen in liquid nitrogen and stored at -80°C.
Serum determinations
Blood was sampled by vein puncture between 09.00 and 11.00 h and the serum stored in aliquots at -20°C. Aldosterone concentration was determined by radioimmunoassay using the aldosterone-MAIA kit from Serono (Nuclear Ibérica, Madrid, Spain). Serum glucose concentration was measured by the glucose oxidase method (Boehringer Mannheim, Germany). The Na+ and K+ concentrations were determined by atomic absorption spectrophotometry (Philips PV9200, Eindhoven, The Netherlands). Osmolality was measured in an Osmomat 30 cryoscopic osmometer (Gonotec, Berlin, Germany).
Brush-border membrane vesicle preparation
Brush-border membrane vesicles (BBMVs) were prepared by a MgCl2 precipitation method (Vázquez et al. 1997). The intestinal scrape was homogenized and MgCl2 was added to the homogenate so as to reach a final concentration of 10 mM. After successive centrifugations, the final pellet containing purified brush-border membranes was resuspended in a medium containing 300 mM mannitol, 0·1 mM MgSO4, 0·41 µM LiN3 and 20 mM Hepes/Tris (pH 7·4), adjusted to a final protein concentration of 15-20 mg ml-1. The protein concentration in the homogenate fraction was 20·1 ± 3·3 mg ml-1 (n = 12), and the overall recovery, calculated as the sum of recoveries of all fractions, was 92·0 ± 3·5 %. The intravesicular volume calculated under equilibrium conditions using 0·1 mM D-glucose (d-Glu) was 0·73 ± 0·16 µl (mg protein)-1 (n = 12).
Basolateral membrane vesicle preparation
Basolateral membrane vesicles (BLMVs) were prepared by the method described by Garriga et al. (1997). Scraping of the mucosa was homogenized and MgCl2 was then added to a final concentration of 8 mM in order to precipitate membranes. Successive centrifugation steps were carried out. After a final centrifugation, the resultant pellet was resuspended in 300-400 µl of a buffer containing 300 mM mannitol, 20 mM Hepes/Tris (pH 7·5), 0·1 mM MgSO4, and 0·41 µM LiN3. The protein concentration in the homogenate fraction was 13·7 ± 1·1 mg ml-1 (n = 12), and the overall recovery was 83·9 ± 3·3 % (n = 6). The intravesicular volume calculated under equilibrium conditions using 1 mM D-Glu was 2·3 ± 0·2 µl (mg protein)-1 (n = 6).
Enzyme and protein determinations
The activity of the ouabain-sensitive Na+-K+-activated ATPase (Na+-K+-ATPase, EC 3.6.1.3) was routinely assayed as a marker enzyme of the basolateral membrane with the method described by Colas & Maroux (1980). The brush-border membrane marker enzyme sucrase (-D-glucohydrolase, EC 3.2.1.48) was assayed with the method of Messer & Dahlqvist (1966). Protein was determined using the Coomassie Brilliant Blue method using bovine serum albumin as standard (Bradford, 1976).
Transport assays
The uptake of -methyl-D-glucoside (Glu1Me) and D-Glu was measured at 37°C by a rapid filtration technique. For determination of initial rates, Glu1Me was incubated with the BBMVs for 10 s (flux was linear for up to 20 s) and D-Glu was incubated with BLMVs for 5 s (flux was linear for up to 10 s). For time course studies vesicles were incubated in the range 5 s to 30 min. For each uptake, 10 µl BBMV or BLMV suspensions (equivalent to 50-150 µg of protein) were rapidly mixed with 40 µl of the incubation medium containing (mM): 100 mannitol, 100 NaCl or KCl, 0·1 MgSO4, 0·1 D-Glu or Glu1Me, 20 Hepes/Tris (pH 7·4); plus 0·41 µM LiN3, and 45 µM valinomycin (only in the case of Glu1Me transport in BBMVs, in order to render the vesicles K+ permeable), and an aliquot of D-[14C]glucose or -methyl-D-[14C]glucoside. At the selected incubation times the uptake was stopped by the addition of 1 ml of an ice-cold stop solution containing (mM): 300 mannitol, 0·2 HgCl2 (only in the case of D-Glu), 0·1 MgSO4, 20 Hepes/Tris (pH 7·4) and 0·41 µM LiN3. The resulting suspension was rapidly filtered under negative pressure through 0·22 µm pore size cellulose nitrate filters (Millipore) and washed with 5 ml stop solution. The filters were dissolved in Biogreen-6 cocktail (Scharlau, Barcelona, Spain) and the radioactivity counted in a Packard TriCarb (model 1500).
The substrate concentrations used for the kinetic analysis of Glu1Me uptake by BBMVs were 0·01, 0·05, 0·075, 0·1, 1, 10, 25 and 75 mM; and for the kinetic analysis of D-Glu uptake by BLMVs were 0·01, 0·5, 1, 5, 15, 50, 100, 150 and 200 mM. The osmolality of intra- and extravesicular media was kept constant at 320 mosmol kg-1 by adjusting the total sugar concentration with mannitol. In conditions where sugar concentration was above 150 mM, osmolality was maintained by reducing mannitol or KCl concentration.
Phlorizin binding measurements
Steady-state phlorizin binding was assayed at 37°C by the method described by Peerce & Clarke (1990) with some modifications. Ten microlitres of BBMV suspensions were rapidly mixed with 40 µl of the incubation medium containing (mM): 100 mannitol, 100 NaCl or KCl, 0·1 MgSO4, Hepes/Tris (pH 7·4); plus 45 µM valinomycin, 50 µM phlorizin, 0·41 µM LiN3 and an aliquot of [3H]phlorizin. At 5 s, the binding process was stopped by addition of 1 ml of an ice-cold stop solution containing (mM): 300 mannitol, 0·1 MgSO4, 20 Hepes/Tris (pH 7·4); and 0·41 µM LiN3. The resulting suspension was rapidly filtered under negative pressure through 0·22 µm pore size cellulose nitrate filters and washed with 5 ml of stop solution. The radioactivity remaining in the filter was determined as described before. Specific [3H]phlorizin binding was calculated by subtracting the non-specific binding (in the presence of K+) from total phlorizin binding (assayed in the presence of Na+). The density of phlorizin binding sites was expressed either as the Bmax, defined as the maximum number of binding sites, or as picomoles of phlorizin bound per milligram of protein at a phlorizin concentration of 50 µM (B50).
Cytochalasin B binding measurements
Steady-state cytochalasin B binding was assayed at 37°C with a method based on that described by Cheeseman & Maenz (1989). Ten microlitres of BBMV suspensions were rapidly mixed with 40 µl of the incubation medium containing (mM): 100 mannitol, 100 L-glucose or D-Glu, 0·1 MgSO4, 20 Hepes/Tris (pH 7·5); plus 0·1 µM cytochalasin B, 0·41 µM LiN3 and an aliquot of [3H]cytochalasin B. At 5 s, the binding process was stopped by addition of 1 ml of an ice-cold stop solution (for composition see 'Phlorizin binding measurements'). The resulting suspension was rapidly filtered under negative pressure through 0·22 µm pore size cellulose nitrate filters and washed with 5 ml of stop solution. The radioactivity remaining in the filter was determined as described before. Total cytochalasin B binding was determined in the medium containing L-glucose, and non-specific binding was determined in the medium containing D-Glu. Specific cytochalasin B binding was calculated by subtracting non-specific from total cytochalasin B binding. The density of cytochalasin B binding sites was expressed as either Bmax or as picomoles of cytochalasin B bound per milligram of protein at a cytochalasin B concentration of 0·1 µM (B0·1).
Chemicals
All unlabelled reagents were obtained from Sigma Chemical Co. except the reagents used to determine enzymatic activity which were from Boehringer Mannheim. D-[U-14C]Glucose (specific activity 251 mCi mmol-1), -methyl-D-[14C]glucoside (specific activity 265 mCi mmol-1), [3H]phlorizin (specific activity 46·4 Ci mmol-1) and [3H]cytochalasin B (specific activity 15 Ci mmol-1) were purchased from New England Nuclear Research Products (Dreieich, Germany). The final activity of labelled substrates in the incubation medium was 0·5-2 µCi ml-1.
Kinetic analysis
Total D-Glu or Glu1Me fluxes from at least three independent experiments were analysed by non-linear regression using the Biosoft Enzfitter program (Cambridge, UK). As errors associated with experimental fluxes were roughly proportional to their values it was considered appropriate to apply a proportional weighting to the data. Kinetic parameter evaluation was made by systematically testing different model equations corresponding to one or two Michaelian components plus a linear non-specific component. The same software was applied to fit the results from probe binding studies and Scatchard transformations.
Statistical analysis
Kinetic parameters and binding measurements were compared according to Student's t test with a level of significance of P < 0·05.
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RESULTS |
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Animal model
Both experimental groups ate similar amounts of wheat and barley (78 ± 8 g (kg body weight)-1 day-1 (n = 12) and 81 ± 7 g (kg body weight)-1 day-1 (n = 12), respectively) and the growth rate during the 2 week adaptation period was also the same. The LS intake induced a secondary hyperaldosteronism that was already maximal after 11 days (Fig. 1) while HS fed birds showed a decrease in serum aldosterone, because the HS diet used contained more NaCl than the commercial diet, in agreement with Árnason & Skadhauge (1991). Table 1 shows that the LS diet increased the serum K+ concentration 28 % and that serum Na+ concentration and osmolality were not modified as has been observed previously in hens (Árnason & Skadhauge, 1991). The table also shows that in the brush-border membrane sucrase activity is not affected by diet, while the Na+-K+-ATPase activity of the basolateral membrane is significantly increased in the rectum.
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Ten-week-old chickens were maintained on the low-NaCl diet for 2 weeks ( | ||
Table 1. Serum variables and enzymatic activity of the intestinal epithelium of chickens adapted to the high-NaCl (HS) and the low-NaCl (LS) diet
| Serum variables | HS diet | LS diet | |
| Na+ (mM) | 135 ± 2 | 136 ± 1 | |
| K+ (mM) | 3·6 ± 0·2 | 4·5 ± 0·2 * | |
| Osmolality (mosmol kg-1) | 312 ± 1 | 310 ± 1 | |
| Glucose (mM) | 14·2 ± 1·3 | 13·9 ± 2·0 | |
| Epithelial enzymes | Segment | HS diet | LS diet |
| -D-Glucohydrolase activity (nmol (mg protein)-1 s-1) | Jejunum | 10·1 ± 0·8 | 9·8 ± 1·2 |
| Ileum | 6·4 ± 0·3 | 7·1 ± 0·9 | |
| Rectum | 6·7 ± 1·4 | 6·3 ± 0·9 | |
| Na+-K+-ATPase activity (nmol (mg protein)-1 s-1) | Jejunum | 1·39 ± 0·13 | 1·43 ± 0·23 |
| Ileum | 1·35 ± 0·03 | 1·34 ± 0·19 | |
| Rectum | 1·11 ± 0·12 | 1·43 ± 0·01 * |
Characterization of the membrane vesicles
The purity of both BBMVs and BLMVs was determined by marker enzyme assays. In the final BBMVs preparation the activity of sucrase was highly enriched and the overall recovery of this enzymatic activity was 87 % (Table 2). The activity of the Na+-K+-ATPase, a marker of the basolateral membrane, was not enriched (0·9 ± 0·1-fold, n = 9). Membrane orientation was studied according to Del Castillo & Robinson (1982), and indicates that 90 ± 2 % of the vesicle population were outside-out oriented. In BLMVs, the enrichment factor for the Na+-K+-ATPase activity was also high and the overall recovery of this marker was 85 % (Table 2). The activity of sucrase was not enriched in BLMVs (1·0 ± 0·2-fold, n = 8). Orientation of BLMVs was routinely checked as described previously (Garriga et al. 1997) demonstrating that the vesicle preparation consists of more than 85 % outside-out vesicles.
Table 2. Recovery and enrichment factor of BBMV and BLMV enzymatic markers
| HS diet | LS diet | |||
| Recovery (%) | BBMVs | BLMVs | BBMVs | BLMVs |
| Jejunum | 89 ± 2 | 87 ± 5 | 87 ± 1 | 83 ± 3 |
| Ileum | 89 ± 2 | 86 ± 1 | 88 ± 4 | 84 ± 2 |
| Rectum | 83 ± 4 | 88 ± 4 | 86 ± 3 | 82 ± 1 |
| Enrichment factor | BBMVs | BLMVs | BBMVs | BLMVs |
| Jejunum | 13·2 ± 1·0 | 10·9 ± 1·1 | 12·8 ± 0·7 | 13·0 ± 0·2 |
| Ileum | 11·8 ± 1·4 | 12·8 ± 0·5 | 13·0 ± 0·1 | 13·2 ± 1·2 |
| Rectum | 12·0 ± 0·9 | 12·3 ± 0·4 | 14·0 ± 1·7 | 10·8 ± 1·5 |
Transport of Glu1Me across BBMVs
In the presence of Na+, BBMVs show an Glu1Me overshoot which is indicative of their capacity to accumulate hexoses against a concentration gradient. There are marked quantitative differences between intestinal regions: the jejunum and the ileum have overshoots that are already maximal after 5 s incubation, giving a 12- to 16-fold D-Glu accumulation over equilibrium values while the rectum shows rather flat overshoots (6-fold accumulation in the HS condition and only 2-fold in the LS fed animals). In the absence of Na+ (K+ present), the uptake had the profile of a passive process, with equilibrium already reached after 30 min. Figure 2 shows that the total initial Glu1Me uptake in the concentration range 0·01-75 mM can be broken down into a linear and a single saturable component, and the calculated kinetic constants are shown in Table 3.
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Glu1Me uptake by BBMVs of jejunum (J), ileum (I) and rectum (R) from chickens adapted to the high- (H) or the low-NaCl (L) diet
Vesicles were incubated (10 s) with | ||
Table 3. Kinetic parameters of Glu1Me uptake in BBMVs of chickens adapted to the HS or the LS diet
| Vmax (pmol (mg protein)-1 s-1) | Km (mM) | Kd (nl (mg protein)-1 s-1) | ||
| Jejunum | HS | 277 ± 78 | 1·7 ± 0·7 | 11·0 ± 0·9 |
| LS | 240 ± 7 | 1·3 ± 0·2 | 14·8 ± 3·3 | |
| Ileum | HS | 225 ± 43 | 1·3 ± 0·1 | 10·8 ± 0·3 |
| LS | 130 ± 15 | 1·0 ± 0·1 | 12·5 ± 0·7 | |
| Rectum | HS | 122 ± 5 | 1·0 ± 0·1 | 19·5 ± 1·4 |
| LS | 47 ± 1 | 1·6 ± 0·1 | 16·8 ± 0·1 |
Glu1Me in concentrations ranging from 0·01 to 75 mM for 10 s. The kinetic parameters were calculated by non-linear analysis (see Methods section). Values are means ± Phlorizin binding measurements
In preliminary experiments the binding kinetics of [3H]phlorizin were studied. Binding experiments carried out in HS chickens showed that the radiolabelled ligand became saturated with an increase in the concentration of [3H]phlorizin (Fig. 3A). The data can be fitted to a rectangular hyperbola:
B = BmaxC/(Ka + C),
where B is bound phlorizin, C is the concentration of phlorizin in the incubation medium and Ka is the apparent affinity constant. Results from the jejunum and the ileum show the same behaviour, with a Bmax of 89 ± 1 pmol (mg protein)-1 while the Bmax in the rectum is lower (51 ± 4 pmol (mg protein)-1). Scatchard plots of binding show that there is a single population of binding sites in the three segments, all having the same Ka (15-18 µM, Fig. 3B).
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Vesicles of jejunum ( | ||
Figure 4 shows the effect of HS or LS diet in specific phlorizin binding to the BBMVs, using a 50 µM phlorizin concentration. Adaptation to LS diet does not affect specific phlorizin binding in the jejunum while in the ileum and the rectum B50 is significantly reduced (P < 0·05). Figure 5 plots the Vmax of Glu1Me transport in all experimental conditions with the phlorizin binding parameter (B50) and the results show that both variables are closely correlated (r = 0·997).
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Vesicles were incubated with 50 µM phlorizin. Values are means ± | ||
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Glu1Me uptake vs. density of phlorizin binding sites in BBMVs
Vmax of | ||
Transport of D-Glu across BLMVs
Incubation of the vesicles with 1 mM D-Glu shows that after 5 s incubation, uptake represents 2-6 % of the amount present in equilibrated vesicles (not shown). Total D-Glu uptake (in nmol (mg protein)-1 s-1) vs. concentration of external D-Glu (ranging from 0·01 to 200 mM) shows the typical profile of a saturable carrier-mediated process (Fig. 6). Total fluxes can be broken down into only one saturable component plus a non-saturable process (Fig. 6), and the calculated kinetic constants are shown in Table 4.
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Vesicles were incubated with D-Glu at concentrations ranging from 0·01 to 200 mM for 5 s. Values are means ± | ||
Table 4. Kinetic parameters of D-Glu uptake in BLMVs of chickens adapted to the HS or the LS diet
| Vmax (pmol (mg protein)-1 s-1) | Km (mM) | Kd (nl (mg protein)-1 s-1) | ||
| Jejunum | HS | 1676 ± 181 | 12·8 ± 0·9 | 20·8 ± 1·8 |
| LS | 1887 ± 95 | 17·6 ± 1·4 | 20·6 ± 1·0 | |
| Ileum | HS | 1773 ± 193 | 18·6 ± 1·4 | 19·2 ± 1·0 |
| LS | 978 ± 163 | 17·5 ± 2·2 | 18·2 ± 0·6 | |
| Rectum | HS | 1242 ± 289 | 15·5 ± 1·4 | 19·4 ± 1·9 |
| LS | 570 ± 71 | 14·7 ± 1·0 | 18·4 ± 0·2 |
Cytochalasin B binding measurements
Figure 7 shows cytochalasin B binding in BLMVs as a function of cytochalasin B concentration. Scatchard plots indicate that in the three segments there is a single population of binding sites, with similar Ka values (0·4-0·5 µM) but differing in their Bmax. Figure 8 shows the effects of diet on specific cytochalasin B binding, using a probe concentration of 0·1 µM (B0·1). Results show that the NaCl content in diet does not affect the density of specific cytochalasin B binding sites in the jejunum while in the ileum and in the rectum of LS-adapted chickens they are significantly reduced (38 and 47 %, respectively). Figure 9 shows that the D-Glu transport variable (Vmax) and the specific binding parameter B0·1 are closely correlated (r = 0·978).
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Vesicles of jejunum ( | ||
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Vesicles of jejunum (J), ileum (I) and rectum (R) from chickens adapted to the high- (H) or the low-NaCl (L) diets, were incubated with 0·1 µM cytochalasin B. Values are means ± | ||
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Vmax of D-Glu and the density of specific 0·1 µM cytochalasin B binding sites (B0·1) are defined by the equation y = 176·6x + 453·4 (r = 0·930). | ||
Effect of 3-oxy-methyl-D-glucose on D-Glu uptake
Figure 10 shows that the non-metabolizable D-Glu analogue 3-oxy-methyl-D-glucose (Glu3Me) at a concentration of 10 mM in the incubation medium has a different inhibitory effect on D-Glu uptake in the jejunum than in the ileum and rectum. Thus, while Glu3Me inhibits the uptake of 0·1 mM D-Glu in the jejunum by 30 %, it has no inhibitory effect in the two other more distal segments. Furthermore, a Glu3Me concentration of 100 mM inhibits D-Glu uptake to the same extent as D-Glu in the jejunum but significantly less in the ileum and the rectum.
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Uptake of 0·1 mM D-Glu (5 s incubation) in the presence of 10 and 100 mM D-Glu and Glu3Me. Results are expressed as the percentage of control fluxes (CTL) and bars show the | ||
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DISCUSSION |
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The intestinal absorption of Na+ in chickens fed either a standard commercial diet or an experimental grain-based high-NaCl (HS) diet, takes place by electroneutral mechanisms and by amiloride-sensitive Na+ channels (Grubb et al. 1987; Skadhauge, 1993). In the small intestine, the proximal caecum and the rectum, but not in the coprodeum, there is a third pathway for Na+ absorption which is cotransported with non-electrolytes such as D-glucose (d-Glu) and certain amino acids (Lind et al. 1980; Ferrer et al. 1994; Amat et al. 1996).
In the brush-border of the chicken rectum, Bindslev et al. (1997) identified a 75 kDa protein that is recognized by antibodies of the sequence of rabbit SGLT1; they also suggest the existence in the rectum of a second high-capacity and low-affinity, SGLT2-like, cotransporter. However, kinetic analyses of uptake across the brush-border using D-Glu, a substrate that can use other SGLT isoforms, indicate that initial fluxes fit best to a single pathway, consistent with the SGLT1 isoform (Planas et al. 1997). Nevertheless, we decided to use Glu1Me as substrate because this glucose analogue is transported by SGLT1 with an apparent Km that is 10-fold that of the SGLT2 isoform (Panayotova-Heiermann et al. 1996). The kinetic analysis confirms there is a single pathway for Glu1Me, with Km values in the range between 1 and 1·7 mM, similar to the values found in isolated enterocytes from the jejunum and the proximal caecum (Ferrer et al. 1986) but lower than those calculated for everted sleeves of chicken intestine (Amat et al. 1996).
Another approach to estimating the number of hexose transporters was to study the binding of labelled phlorizin to quantify the apical 'phlorizin-sensitive' hexose uptake. In HS birds the maximal binding capacities (Bmax) in the jejunum and ileum were similar (mean value of 89 pmol (mg protein)-1) and higher than that of the rectum (51 pmol (mg protein)-1). These figures are in the range of those reported by Ishikawa et al. (1997) for vesicles from the rat small intestine. The Scatchard plot gives a straight line in the three segments, consistent with the presence of a single transporter. Phlorizin binding was affected by the intestinal region and diet, and the changes observed were well correlated with the maximal transport capacity. The fact that the interception in the y-axis does not differ from zero (see Fig. 5) indicates that the differences in Vmax obtained in the experimental groups, according to diet and intestinal region, can be attributed to variation in density of the phlorizin binding sites.
Our results show that the increase in the number of transporters fully accounts for the increase in the transport rates, similar to the observations of Diamond & Karasov (1984) in mice adapted to diets containing varying amounts of carbohydrates. Calculation of the turnover for one phlorizin binding site at 37°C from the slope of Fig. 5 results in a mean value of 180 min-1 which is lower than the figures calculated by Wright & Peerce (1985) for the rabbit glucose transporter at room temperature.
The kinetics of the basolateral hexose carrier in more distal segments of the chicken have not been studied before but it has been predicted that their transport capacity would be much lower than that of the jejunum (Ferrer et al. 1994). The kinetic study of the basolateral GLUT2 transporter confirms that it is a high capacity transport system with relatively low affinity for D-Glu. It is worth noting that in the rat jejunum Cheeseman (1992) calculated a Vmax for D-Glu of about 3600 pmol (mg protein)-1 s-1, a figure that is higher than the value found in the chicken taking into account that the rat experiments were carried out at 22°C.
The study of the basolateral GLUT2 transporters showed some discrepancy between the results of kinetic and the binding experiments using labelled cytochalasin B. Thus, the kinetic results clearly show that the HS jejunum and the HS ileum have the same Vmax, and both are higher than the Vmax value in the HS rectum. However, the binding parameter B0·1 is higher in the jejunum than in the ileum which in turn is higher than in the rectum. This would indicate that the GLUT2 turnover in the ileum (calculated to be 16·3 × 103 min-1) is higher than in the jejunum (12·4 × 103 min-1). The effects of LS adaptation in the basolateral membrane are similar to the results in the brush- border, because low-NaCl intake has no effect on the jejunum but induces a significant reduction in both Vmax and cytochalasin B binding in the ileum and the rectum, indicating that in the more distal regions LS diet reduces the incorporation of basolateral hexose transporters.
The finding that the basolateral membrane of the ileum and the rectum show a relatively high Vmax for D-Glu and high cytochalasin B binding (both indicative of a high number of basolateral transporters) was unexpected. Both regions were believed to have low hexose flux permeability for two reasons: first, they had higher hexose accumulation ratios at steady state (23-fold in the ileum and 25-fold in the rectum) compared with the jejunum (8-fold) because the capacity to accumulate a substrate depends, among other factors, on its efflux permeability; second, in isolated enterocytes we had measured lower influx rates for hexoses in ileum and rectum than in the jejunum (Ferrer et al. 1994). We were aware, however, that previous conclusions about the characteristics of the basolateral hexose transport had been obtained indirectly, using the D-Glu analogues 3-oxy-methyl-D- glucose (Glu3Me) and 2-deoxy-glucose, while flux experiments for the present kinetic study have been done using D-Glu as substrate. For this reason we wanted to check if Glu3Me behaves like D-Glu in the basolateral membrane and to this end we carried out inhibition studies comparing the effects of both hexoses on D-Glu uptake. The results support the view that the affinity of Glu3Me for the GLUT2 transporter is higher in the jejunum than in the ileum and the rectum, and this explains why Glu3Me can reach a cytosol concentration at steady state that is much higher in the ileum and rectum than in the jejunum. The finding of regional differences in GLUT2 Km values for the D-Glu analogue indicates that the kinetic properties of the GLUT2 isoform vary along the intestine.
Low-Na+ adaptation has no effect on hexose uptake by the jejunum and the effect on the ileum, albeit significant, is small. However, in the rectum, the apical Vmax is reduced by 71 % and basolateral Vmax by 54 %. Assuming a luminal D-Glu concentration in the rectum of 1·5 mM and a Km for D-Glu of 1 mM (Planas et al. 1997), the apical flux would drop from 101 to 42 pmol (mg protein)-1 s-1. However, the actual flux would be lower because LS adaptation decreases the electrical gradient across the membrane (Jaso et al. 1995). In the basolateral membrane, the calculated fluxes for a D-Glu concentration of 14 mM in the fluid bathing the membrane are 552 pmol (mg protein)-1 s-1 in HS- and 253 pmol (mg protein)-1 s-1 in LS-adapted animals. This means that the basolateral membrane retains a significant capacity to take up D-Glu, which will eventually fulfil the needs for metabolic fuel from the blood compartment.
A final point is the nature of the signal that controls the whole process. In the chicken we have demonstrated that dietary lysine supplementation results in an increased transport capacity as a result of upregulation of existing carriers (Torras-Llort et al. 1998). Similar observations have been made in mice fed varying amounts of carbohydrates (Diamond & Karasov, 1984). In these examples it is the changes in nutrient availability that regulates transport. However, in the present study both groups of animals eat the same kind and amount of food and therefore the luminal concentration of D-Glu is presumably the same, which supports the view that the signal controlling the change in transport capacity is not of lumenal origin. Aldosterone seems likely to be the agent that controls induction of both - and 
-subunits of the epithelial Na+ channel (Goldstein et al. 1997). It stimulates NHE2 incorporation to the membrane (Donowitz et al. 1998). It also stimulates Na+-K+-ATPase activity (Table 1; Skadhauge, 1993). Thus it would appear a good candidate to account for the effects observed in sugar movements across the epithelium. There are, however, unanswered questions regarding the level and specificity of the regulatory process.
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C. Garriga was a recipient of a Formació d'Investigadors grant from Generalitat de Catalunya. We thank Dr M. A. Navarro (Hospital de Bellvitge, L'Hospitalet) and Dr M. J. Jaso for their help in serum determinations, and M. Ferrándiz for excellent technical assistance. This work was supported by Ministerio de Educación y Cultura, Spain.
Corresponding author
M. Moretó: Departament de Fisiologia-Divisió IV, Facultat de Farmàcia, Universitat de Barcelona, Av. Joan XXIII, s/n, E-08028 Barcelona, Spain.
Email: mmoreto{at}far.ub.es
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). There is already a significant increase (P < 0·05) after 5 days of low-NaCl feeding. The symbol (
) refers to the serum aldosterone concentration at day 14 in birds fed the high-NaCl diet (39 ± 5 pg ml-1). This is significantly (P < 0·05) lower than at day 0 (70 ± 9 pg ml-1). Each point is the mean ± 
) and rectum (
) were incubated for 5 s with 0·1-100 µM phlorizin. A, saturation curves. The constants calculated for the jejunum and ileum were similar and were thus plotted together; the affinity constant (Ka) was 18·1 ± 0·03 µM and the maximum number of binding sites (Bmax) was 88·6 ± 0·1 pmol (mg protein)-1. In the rectum, Ka was 14·9 ± 2·1 µM, and Bmax 51·0 ± 4·2 pmol (mg protein)-1. B, Scatchard plot of data in A. Regression coefficients (r > 0·949) support the view that the three segments have a single population of binding sites.