Siegfried Wolffram

Kinetics of D-glucose and L-leucine transport into sheep and pig intestinal brush border membrane vesicles.

The kinetic parameters (Vmax, Kt) of Na+-dependent D-glucose transport into brush border membrane vesicles (BBMV) from sheep and pig jejunum were determined. Due to the fermentation of ingested carbohydrates in the rumen the small intestine of ruminants (sheep) has to absorb much less glucose than the small intestine of monogastric omnivores (pigs) or herbivores. Kinetic analysis of the concentration dependence of D-glucose transport revealed a ten-fold smaller Vmax value combined with a five times lower Kt value in sheep BBMV compared with pig BBMV. The Vmax value for L-leucine transport did not differ between the two species investigated, whereas the Kt value in the sheep exceeded that in the pig. It is concluded from these results that the mechanism for Na+-dependent D-glucose transport in ruminants is adapted to the small amounts of carbohydrates reaching the small intestine.

Uptake of selenate and selenite by isolated intestinal brush border membrane vesicles from pig, sheep, and rat

Selenate and selenite uptakes by isolated intestinal brush border membrane vesicles (BBMV) from pig, sheep, and rat were investigated. Selenate uptake into jejunal and ileal, but not duodenal, BBMV from pig was stimulated by an inwardly directed transmembrane Na+ gradient (Naout+>Nain+). Selenate transport into rat ileal and sheep jejunal BBMV was also enhanced in the presence of a Na+ gradient. Unlike selenate uptake, selenite uptake was not Na+ dependent, neither in pig small intestine nor in sheep jejunum and rat ileum. Uptake of selenate represented real uptake into the vesicular lumen, whereas selenite uptake was a result of an extensive binding of75Se to the membranes. Thiosulfate at a 250-fold concentration of selenate completely inhibited Na+-dependent selenate uptake into pig jejunal BBMV. Furthermore, Na+-dependent sulfate uptake was totally inhibited in the presence of a 250-fold selenate concentration. The results clearly show that selenate transport across the BBM of pig jejunum and ileum, sheep jejunum, and rat ileum is partially energized by a transmembrane Na+ gradient. Moreover, it is concluded from the results that there exists a common transport mechanism for sulfate and selenate in the BBM. The extensive binding of75Se from75Se-labeled selenite to the membranes could be from a spontaneous reaction of selenite with membrane-associated SH groups.

Stimulation of mucosal uptake of selenium from selenite by some thiols at various sites of rat intestine

The influence of several thiols (conc. 1 mmol/L) on mucosal uptake of75Se from75Se-labeled selenite (conc. 10 μmol/L) across the brush border of rat jejunum and cecum was investigated in vitro using a short-term uptake technique.l-Cysteine (l-Cys) stimulated75Se uptake in the mid- and distal jejunum and cecum, but not in the proximal jejunum. The effect was maximal in the distal jejunum.d-Cys was less effective in the jejunum and similarly effective in the cecum.l-Leucine (l-Leu) andl-glutamic acid significantly reduced the stimulatory effect ofl-Cys on Se uptake in the distal jejunum, whereas the respective effect ofd-Cys was not diminished byl-Leu. Cysteamine stimulated mucosal75Se uptake at all intestinal sites tested, whereas the effect of mercaptopyruvate was restricted to the distal jejunum. Thioglycolate also enhanced75Se uptake in the distal jejunum. The stimulatory effects ofl-Cys, mercaptopyruvate, and thiologlycolate were Na+-dependent, whereas the effect of cysteamine also occurred in the absence of Na+. Mercaptosuccinate,d-penicillamine, ergothioneine, and thiosulfate did not enhance mucosa75Se uptake. It is concluded from these findings that the reaction of some thiols with selenite results in Se compounds that are rapidly absorbed by the intestinal epithelium through various Na+-dependent and Na+-independent, mechanisms. The high bioavailability of Se from selenite found by others might thus be the result of the presence of thiols in the gastrointestinal tract.

Transport of selenate and selenite across the brush border membrane of rat and sheep small intestine

Mucosal uptake of75Se-labeled selenate and selenite across the brush border was investigated in sheep and rat small intestine, using 3-min mucosal exposures. Uptake of selenate and selenite occurred faster in rat than in sheep small intestine. With the exception of sheep duodenum, mucosal selenate uptake was Na+-dependent in sheep and rat small intestine. Mucosal uptake of selenite across the brush border was Na+-dependent only in sheep midjejunum, whereas it was Na+-independent in sheep duodenum and ileum and the rat whole small intestine. Various anions inhibited selenate transport in the presence of Na+ in sheep midjejunum in the order S2O22- = CrO42- > MoO42- and in rat ileum in the order CrO42- = S2O32- > SC42- > MoO42-. Thiosulfate also inhibited mucosal selenite uptake in the presence of Na+ in sheep midjejunum. Preincubation of rat ileum with glutathione (GSH) enhanced mucosal selenite uptake, whereas selenate uptake remained unaffected.These results indicate that selenate transport across the brush border membrane is energized in part by the Na+-gradient. Moreover, the Na+-dependent transport mechanism for the Se salts apparently has an affinity for other anions (S2O32-, SO42-, CrO42-, MOo42-). The findings further indicate that intracellular GSH plays a role in the absorption of selenite, probably by an increase of intracellular selenite metabolism. The Na+-independent mucosal uptake of selenate and selenite probably represents diffusion.

Stimulation of mucosal uptake of selenium from selenite byl-cysteine in sheep small intestine

The influence of cysteine (Cys) on mucosal uptake of75Se-labeled selenite in sheep midjejunum was investigated using a short-term uptake technique.l-Cys (concn.: 1.0 mmol/L) significantly stimulated uptake of Se from selenite (concn.: 10 μmol/L). The stimulatory effect ofl-Cys on mucosal uptake of Se from selenite was Na+—and pH-dependent. In the absence of Na+, or at an acidic pH (5.0), the stimulatory effect ofl-Cys was abolished.l-alanine andl-lysine, but notl-glutamic acid inhibited uptake of Se from selenite in the presence ofl-Cys. Preincubation of mucosal preparations with 10 mmol/Ll-Cys produced enhanced mucosal uptake of Se from selenite.It is concluded from these results thatl-Cys stimulates absorption of Se from selenite probably by generation of selenodicysteine and maybe cysteine selenopersulfide that are subsequently transported across the intestinal brush border membrane by Na+-dependent amino acid carriers. Furthermore, intracellular generation of selenodicysteine might contribute to the uptake of Se from selenite by maintaining the concentration gradient for diffusive uptake of selenite.

Kinetics of d-glucose transport across the intestinal brush-border membrane of the cat

1. D-glucose transport across the intestinal brush-border membrane of the cat, a carnivorous animal, was investigated using isolated brush-border membrane vesicles (BBMV). Kinetic experiments were performed under zero-trans conditions (initial [Na+]in and [Gluc]in = O) with the transmembrane electrical potential difference clamped to zero. 2. D-glucose uptake by the BBMV was strongly stimulated by an inwardly directed Na+-gradient. Uptake under Na+-free conditions seemed to occur by simple diffusion. 3. The apparent kinetic constants (Vmax, Km) of Na+-dependent D-glucose transport were computed by forcing initial uptake rates at 0.002-10.0 mmol/l D-glucose to either a Michaelis-Menten type equation with a single or with two carrier-mediated components. 4. Best fit of the experimental data was obtained with the two-component model indicating the existence of two Na+-dependent carrier-mediated mechanisms. System 1 and system 2 differ with respect to the transport velocity as well as the substrate affinity constants with Vmax being 2.5-fold and Km being 5-fold higher for system 1 compared with system 2.

Transport of citrate across the brush border and basolateral membrane of rat small intestine.

It was the aim of the present study to investigate the transport of tricarboxylates (citrate, tricarballylate) across the basolateral membrane (BLM) of the small intestine. Experiments were performed using BLM vesicles isolated from the jejunum of rats. For comparison, some experiments with brush border membrane (BBM) vesicles were also performed. Finally, transfer of citrate and tricarballylate across the intestinal wall was investigated using sacs of everted small intestine. Uptake of citrate by BBM vesicles occurs by a Na+ gradient-driven transport mechanism specific for tri- and dicarboxylates. The partially protonated forms of citrate seem to be much better transported than the completely dissociated form, since lowering the extravesicular pH from 7.8 to 5.6 resulted in a marked stimulation of Na(+)-dependent citrate uptake. In contrast to citrate uptake across the BBM, uptake of citrate across the BLM was neither influenced by Na+ nor by pH changes. Neither structurally related tri- and dicarboxylates (tricarballylate, succinate) nor other organic and inorganic anions (e.g. lactate, p-aminohippurate, sulfate, chloride, bicarbonate) significantly influenced citrate uptake by BLM vesicles under cis-conditions. Uptake of citrate as a function of the extravesicular substrate concentration was linear over a concentration range from 0.1 to 10 mmol/l. Thus, citrate uptake under these conditions seems to be Na(+)-independent and not to be mediated by a carrier. However, preloading the BLMV with citrate clearly trans-stimulated the uptake of citrate and tricarballylate, respectively. Furthermore, citrate significantly inhibited tricarballylate uptake into BLMV preloaded with citrate. These results indicate uptake of tricarboxylates across the BLM by an exchange mechanism. Using sacs of everted small intestine, no transfer of intact citrate against a concentration gradient occurred, but some evidence for metabolization of citrate within the intestinal wall was obtained. In contrast, the non-metabolizable tricarboxylate tricarballylate was significantly accumulated in the serosal compartment of everted intestinal sacs.

Effects of glutathione and of cysteine on intestinal absorption of selenium from selenite

The influence of glutathione (1 mmol/L) (GSH) on in vitro mucosal uptake and in vivo absorption of75Se-labeled selenite (10 μmol/L) was investigated in rat jejunum. For comparison, the effect ofl-cysteine (1 mmol/L) on in vivo absorption of75Se-labeled selenite was also studied. In the in vitro, uptake experiments, only the mucosal surface was exposed to the incubation medium for 3 min. For the in vivo experiments, a luminal perfusion technique was employed. GSH inhibited in vitro mucosal Se uptake, whereas absorption in vivo was stimulated by GSH.l-Cysteine also stimulated in vivo Se absorption, confirming former in vitro mucosal uptake experiments. Thus, unlikel-cysteine, GSH affected in vitro and in vivo absorption of Se from selenite differently. Enzymatic cleavage of products of the reaction of selenite with GSH occuring more efficiently under in vivo than in vitro conditions may be a prerequisite for the stimulatory effect of GSH on Se absorption. This apparently does not apply to the stimulatory effect of cysteine. Since, GSH occurs in the intestinal lumen under physiological conditions, it may contribute to the high bioavailability of Se from selenite.

Colonic Absorption and Bioavailability of the Pentapeptide Metkephamid in the Rat

The concept of delivering systemically active peptide drugs to the colon in order to improve their oral absorption requires reasonable peptide permeability of the large intestinal wall and stability against the activity of the colonic microflora. In addition, the role of hepatic extraction needs to be addressed. In this study the absorption of the pentapeptide metkephamid following single pass perfusion of rat ascending colon was investigated by monitoring its disappearance from the large intestine and simultaneous appearance in the portal vein, the hepatic vein and the aorta. In addition its stability against colonic microflora was tested in vitro using pig caecal contents. Metkephamid was absorbed from the large intestine and appeared in the blood circulation; peptide concentrations in the portal vein increased over-proportionally with increasing perfusate concentrations (0.1 − 4.6 mmol/L) from 0.19 µg/mL ± 0.12 (SD, n = 7) to 31.6 µg/mL + 20.65 (SD, n = 4), respectively, and thus suggesting a saturable transport or metabolism. Concentrations in the hepatic vein were significantly lower than in the portal vein, hepatic extraction ratios were 0.35 ± 0.14, 0.61 ± 0.18 and 0.62 ± 0.28 (SD, n = 4) for 0.1, 0.5 and 1.0 mM metkephamid perfusate concentrations, respectively. In the anaerobic colon metabolism model the degradation half-life of the peptide was 14.9 hours, thus, indicating relative stability in the bacterial environment of the colon. The results of the present study encourage further investigations on colonic delivery of peptide drugs.

Influence of nitrate and nitrite on electrolyte transport by the rat small and large intestine

1. The influence of nitrate and nitrite on net absorption of electrolytes (Na+, K+, Cl-) and water from ligated loops was studied at various intestinal sites in rats. 2. Nitrate strikingly reduced Cl- absorption in rat proximal and distal colon, whereas Na+ absorption was reduced only moderately. Nitrite also reduced Cl- absorption in the colon. 3. Nitrate showed no significant effect on electrolyte absorption in the small intestine. 4. The results suggest that Cl-/HCO3- on exchange is the major route of Cl- absorption in the colon, whereas this mechanism seems not to be of importance for Cl- absorption by the small intestine.