Fe2+ uptake by mouse intestinal mucosa in vivo and by isolated intestinal brush-border membrane vesicles

In vivo kinetics of mucosal uptake of luminal ⁵⁹Fe²⁺ by tied segments of normal mouse duodenum are characterised by a K_m of approx. 100 μM and a V_max of approx. 9 pmol/min per mg wet weight of intestine. These values were determined at pH 7.25 in the presence of excess sodium ascorbate. Studies with luminal Fe²⁺ concentrations of 100 μM reveal: (1) uptake is relatively independent of ascorbate: Fe ratio and luminal pH and (2) uptake is potently inhibited by 1 mM Co²⁺ or Mn²⁺ and large luminal NaCl concentrations but not by Ca²⁺. 3 days of hypoxia (0.5 atmospheres) yields no significant increase in subsequent total mucosal uptake by in vivo tied segments while uptake is significantly reduced by semi-starvation. Quantitative comparison of in vivo mucosal uptake with subsequent determination of isolated brush-border membrane ⁵⁹Fe²⁺ transport in individual mice reveals a positive correlation (P < 0.01) between the two parameters. These results, in conjunction with studies of isolated mouse duodenal brush-border membrane (Simpson, R.J. and Peters, T.J. (1985) Biochim. Biophys. Acta, 814, 381–388 and (1986) Biochim. Biophys. Acta 856, 109–114) suggest that the Fe²⁺ transport properties of isolated brush-border membrane are quantitatively adequate to explain in vivo mucosal uptake in normal and hypoxic mice at Fe²⁺ concentrations up to 100 μM.     

Studies of Fe3+ transport across isolated intestinal brush-border membrane of the mouse

Mouse intestinal brush-border membrane vesicles take up iron from media containing 59Fe3 +-nitrilotriacetic acid. The iron uptake by the vesicles represents accumulation of iron which relates to an osmotically active space. Uptake is linearly related to vesicle protein concentration and is inhibited by low incubation temperature and low medium free Fe3+ concentrations. Experiments with the lipid soluble iron ligand 8-hydroxyquinoline and with Triton X-100 imply that the uptake is rate limited by membrane transport.     

Fe2+ uptake by intestinal brush-border membrane vesicles from normal and hypoxic mice

Fe2+ uptake by mouse intestinal brush-border membrane vesicles consists of two components: a rapid, high affinity (Kd less than 1 microM), low capacity binding (less than 2 nmol/mg protein), presumably to the outside of the vesicles, and a second, large capacity component with an initial rate showing a hyperbolic dependence on medium Fe2+ (Km 35-90 microM). The latter, predominant process is relatively independent of medium ascorbate: Fe2+ ratio, is inhibited by Co2+ and Mn2+ but varies greatly from one membrane preparation to another. This component is strongly inhibited by large extravesicular NaCl and KCl concentrations and may represent transport into the vesicles. No significant change in uptake could be observed in vesicles prepared from hypoxic mice.     

Cholate-soluble and -insoluble iron binding components of rabbit duodenal brush-border membrane. Relevance to Fe2+ uptake by brush-border membrane vesicles

Fe2+ uptake by brush-border membrane vesicles from rabbit duodenum has been investigated and found to show similar qualitative properties to those previously demonstrated with mouse proximal intestine brush-border membrane vesicles (Simpson, R.J. and Peters, T.J. (1986) Biochim. Biophys. Acta 856, 109-114). In particular, a relatively low affinity (Km(app) approx. 83 microM), NaCl and pH sensitive transport component is present. The disruption of 59Fe2+-laden vesicles with sodium cholate, followed by gel filtration or centrifugal analysis reveals that cholate insoluble material (Mr greater than 10(6)) is the major destination for 59Fe2+ taken up by intact vesicles. Analysis of cholate extracts for Fe2+ binding ability reveals a single high-capacity (49.8 +/- 15.6 nmol/mg vesicle protein (S.E., n = 3)), high-affinity (Kd(app) less than 5 microM) binding component with an Mr equivalent to approx. 10(4) on gel filtration in the presence of cholate. This binding component is extracted into chloroform/methanol (2:1, v/v) is relatively heat and protease resistant and thus appears to be a lipid.     

pH-sensitive transport of Fe2+ across purified brush-border membrane from mouse intestine

Recent studies of Fe2+ uptake by mouse proximal intestine brush-border membrane vesicles revealed low-affinity, NaCl-sensitive and high-affinity, NaCl-insensitive, components of uptake (Simpson, R.J. and Peters, T.J. (1985) Biochim. Biophys. Acta 814, 381-388). In this study, the former component is demonstrated to show a strong pH dependence with an optimum of pH 6.8-6.9. Studies at pH 6.5, where the low affinity component is inhibited by more than 25-fold compared with pH 7.2, suggest that the pH-sensitive component represents transport across the brush-border membrane followed by intravesicular binding. Cholate extracts of brush-border membrane vesicles contain pH- and NaCl-sensitive Fe2+ binding moieties which may be involved in the transfer of Fe2+ across the intestinal brush-border membrane and subsequent binding inside the vesicles. Fe2+ uptake by brush-border membrane vesicles from the duodenum of hypoxic mice is higher than uptake by vesicles from control-fed animals, suggesting the existence of a regulable brush-border membrane Fe2+ carrier.     

Membrane-potential-dependent uptake of tryptamine by rat intestinal brush-border membrane vesicles.

The effect of membrane potential on the uptake of tryptamine, an organic cation, by rat intestinal brush-border membrane vesicles was studied. In the presence of an outwardly directed H(+)-gradient, the initial uptake of tryptamine was stimulated remarkably and the overshoot phenomenon was observed. In contrast, the uptake was depressed by an inwardly-directed H(+)-gradient. The effect of H(+)-gradient on the uptake of tryptamine was maintained in the presence of FCCP, whereas it vanished when voltage-clamped vesicles were used. Moreover, the uptake of tryptamine was linearly augmented with increase of the valinomycin-induced inside-negative K+ diffusion potential. These results suggest that tryptamine is taken up into intestinal brush-border membrane vesicles depends upon the ionic diffusion potential. The effect of several indole derivatives and amine compounds on the uptake of tryptamine was also examined. The uptake of tryptamine was inhibited by all amine compounds used, but anionic and zwitterionic compounds had no effect, suggesting that these amines interact on brush-border membrane and cause an inhibitory effect.     

D-glucose uptake in intestinal brush-border membrane vesicles of rachitic rats

We have previously reported the metabolic consequences of feeding rats Steenbock and Black's rickets-inducing diet, deficient in vitamin D and with an altered Ca/P ratio. Using isolated brush-border membrane vesicles prepared from the jejunum, ileum and duodenum of control and rachitic rats, we have demonstrated a marked decrease of Na+-dependent D-glucose uptake at jejunum-ileum level of rachitic rats. At duodenum level Na+-dependent D-glucose transport was not influenced by rickets. A lack of any significant difference between the two animal groups was observed studying the facilitated transport of D-glucose, the diffusion of L-glucose and the Na+-dependent uptake of phenylalanine and aspartate.     

Aboral changes in D-glucose transport by human intestinal brush-border membrane vesicles.

D-Glucose transport was investigated in isolated brush-border membrane vesicles from human small intestine. Characteristics of D-glucose transport from the jejunum were compared with that in the mid and terminal ileum. Jejunal and mid-ileal D-glucose transport was Na+-dependent and electrogenic. The transient overshoot of jejunal D-glucose transport was significantly greater than corresponding values in mid-ileum. The terminal ileum did not exhibit Na+-dependent D-glucose transport, but did exhibit Na+-dependent taurocholate transport. Na+-glucose co-transport activity as measured by tracer-exchange experiments was greatest in the jejunum, and diminished aborally. We conclude that D-glucose transport in man is Na+-dependent and electrogenic in the proximal intestine and directly related to the activity of D-glucose-Na+ transporters present in the brush-border membranes. D-Glucose transport in the terminal ileum resembles colonic transport of D-glucose.     

Anion antiport mechanism is involved in transport of lactic acid across intestinal epithelial brush-border membrane

Intestinal epithelial membrane transport of L-lactic acid was characterized using rabbit jejunal brush-border membrane vesicles (BBMVs). The uptake of L-[(14)C]lactic acid by BBMVs showed an overshoot phenomenon in the presence of outward-directed bicarbonate and/or inward-directed proton gradients. Kinetic analysis of L-[(14)C]lactic acid uptake revealed the involvement of two saturable processes in the presence of both proton and bicarbonate gradients. An arginyl residue-modifying agent, phenylglyoxal, inhibited L-[(14)C]lactic acid transport by the proton cotransporter, but not by the anion antiporter. The initial uptakes of L-[(14)C]lactic acid which are driven by bicarbonate ion and proton gradients were inhibited commonly by monocarboxylic acids and selectively by anion exchange inhibitor 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid and protonophore carbonylcyanide p-trifluoromethoxyphenylhydrazone, respectively. These observations demonstrate that L-lactic acid is transported across the intestinal brush-border membrane by multiple mechanisms, including an anion antiporter and a previously known proton cotransporter.     

Fe3+ transport by brush-border membrane vesicles isolated from normal and hypoxic mouse duodenum and ileum

Studies of 59Fe3+ uptake by brush-border membrane vesicles prepared from mouse duodenum have indicated that uptake represents transport across the brush-border membrane which is rate-limited by the membrane-transfer step (Simpson, R.J. and Peters, T.J. (1984) Biochim. Biophys. Acta 772, 220-226). Further studies presented here reveal that the uptake rate represents the net influx rate for Fe3+ and is independent of Na+ in the medium and of the method of vesicle preparation. Uptake by brush-border membrane vesicles prepared from mouse distal ileum also represents predominantly transport and is higher than that observed with duodenal brush-border membrane vesicles. Studies of the initial uptake rate by vesicles prepared from normal and hypoxic mouse intestine demonstrated an increase in Fe3+ transport in duodenal vesicles only.     

L-Carnitine transport in mouse renal and intestinal brush-border and basolateral membrane vesicles.

We characterized the uptake of carnitine in brush-border membrane (BBM) and basolateral membrane (BLM) vesicles, isolated from mouse kidney and intestine. In kidney, carnitine uptake was Na(+)-dependent, showed a definite overshoot and was saturable for both membranes, but for intestine, it was Na(+)-dependent only in BLM. The uptake was temperature-dependent in BLM of both kidney and intestine. The BBM transporter in kidney had a high affinity for carnitine: apparent K(m)=18.7 microM; V(max)=7.85 pmol/mg protein/s. In kidney BLM, similar characteristics were obtained: apparent K(m)=11.5 microM and V(max)=3.76 pmol/mg protein/s. The carnitine uptake by both membranes was not affected within the physiological pH 6.5-8.5. Tetraethylammonium, verapamil, valproate and pyrilamine significantly inhibited the carnitine uptake by BBM but not by BLM. By Western blot analysis, the OCTN2 (a Na(+)-dependent high-affinity carnitine transporter) was localized in the kidney BBM, and not in BLM. Strong OCTN2 expression was observed in kidney and skeletal muscle, with no expression in intestine in accordance with our functional study. We conclude that different polarized carnitine transporters exist in kidney BBM and BLM. L-Carnitine uptake by mouse renal BBM vesicles involves a carrier-mediated system that is Na(+)-dependent and is inhibited significantly by specific drugs. The BBM transporter is likely to be OCTN2 as indicated by a strong reactivity with the anti-OCTN2 polyclonal antibody.     

Characterization of sodium-dependent nucleoside transport in rabbit intestinal brush-border membrane vesicles

The characteristics of uridine transport were studied in rabbit intestinal brush-border membrane vesicles. Uridine was taken up into an osmotically active space in the absence of metabolism and there was no binding of uridine to the membrane vesicles. Uridine uptake was markedly enhanced by sodium, but showed no significant stimulation by other monovalent cations tested. Kinetic analysis of the sodium-dependent component of uridine flux indicated a single system obeying Michaelis-Menten kinetics (Km value of 6.4 +/- 1.4 microM with a Vmax of 9.1 +/- 3.6 pmol/mg protein per s as measured under zero-trans conditions with a 100 mM NaCl gradient at 24 degrees C). A variety of purine and pyrimidine nucleosides were able to inhibit sodium-dependent uridine transport, suggesting that these nucleosides are also permeants for the same system. Consistent with this suggestion was the finding that these nucleosides also stimulated uridine efflux from the brush-border membrane vesicles. The sodium: uridine coupling stoichiometry was found to be 1:1 as measured by the activation method. From these results it is concluded that a broad specificity sodium-dependent nucleoside transporter is present at the brush-border membrane surface of rabbit enterocytes.     

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.     

Studies of the kinetics of Na+ gradient-coupled glucose transport as found in brush-border membrane vesicles from rabbit jejunum

The Na+-dependent D-glucose transport reaction in rabbit jejunal brush-border vesicles was studied. Initial rate data were obtained by fitting a polynomial equation to progress curves at different D-glucose concentrations and extracting the slope of the tangent at zero-time. Kinetic replots of the initial rate values produced biphasic Hofstee patterns indicative of two pathways for transport distinguished by their Km values for glucose. Neither was dependent on the presence of a membrane potential. Both were dependent on Na+ and both were inhibited by phlorizin. Increasing external sodium was found to elevate the apparent Vmax for both pathways. Internal sodium was inhibitory. Pulsed progress curve analysis indicated that the effect of internal sodium was best characterized as carrier sequestration by a sodium-carrier binary complex. Inhibition by internal sodium was completely reversed by the presence, internally, of D-glucose. The presence of two pathways and the kinetic constants for these pathways do not agree with the conclusions of Hopfer and Groseclose (1980) J. Biol. Chem. 255, 4453-4462). Experiments are presented which bear on the reason for the disagreement.     

Significance of non-esterified fatty acids in iron uptake by intestinal brush-border membrane vesicles

Iron uptake from Fe/ascorbate by mouse brush-border membrane vesicles is not greatly inhibited by prior treatment with a variety of protein-modification reagents or heat. Non-esterified fatty acid levels in mouse proximal small intestine brush-border membrane vesicles show a close positive correlation with initial Fe uptake rates. Loading of rabbit duodenal brush-border membrane vesicles with oleic acid increases Fe uptake. Depletion of mouse brush-border membrane vesicle fatty acids by incubation with bovine serum albumin reduces Fe uptake. Iron uptake by vesicles from Fe/ascorbate is enhanced in an O2-free atmosphere. Iron uptake from Fe/ascorbate and Fe3+-nitrilotriacetate (Fe3+-NTA) were closely correlated. Incorporation of oleic acid into phosphatidylcholine/cholesterol (4:1) liposomes leads to greatly increased permeability to Yb3+, Tb3+, Fe2+/Fe3+ and Co2+. Ca2+ and Mg2+ are also transported by oleic acid-containing liposomes, but at much lower rates than transition and lanthanide metal ions. Fe3+ transport by various non-esterified fatty acids was highest with unsaturated acids. The maximal transport rate by saturated fatty acids was noted with chain length C14-16. It is suggested that Fe transport can be mediated by formation of Fe3+ (fatty acid)3 complexes.     

Characteristics of glutamic acid transport by rabbit intestinal brush-border membrane vesicles. Effects of Na+-, K+-and H+-gradients

In the presence of a Na⁺-gradient (out > in), l-glutamic acid and l-and d-aspartic acids were equally well concentrated inside the vesicles, while no transport above simple diffusion levels was seen by replacement of Na⁺ by K⁺. Equilibrium uptake values were found inversely proportional to the medium osmolarity, thus demonstrating uptake into an osmotically sensitive intravesicular space. The extrapolation of these lines to infinite medium osmolarity (zero space) showed only a small binding component in acidic amino-acid transport. When the same experiment was performed at saturating substrate concentrations, linear relationships extrapolating through the origin but showing smaller slope values were recorded, thus indicating that the binding component could be more important than suspected above. However, binding to the membrane was neglected in our studies as it was absent from initial rate measurements. Na⁺-dependent uphill transport of l-glutamic acid was stimulated by K⁺ present on the intravesicular side only but maximal stimulation was recorded under conditions of an outward K⁺-gradient (in > out). Quantitative and qualitative differences in the K⁺ effect were noted between pH 6.0 and 8.0. Initial uptake rates showed pH dependency in $\text{Na}{}^{\mathrm{+}}\text{-(out > in) + K}{}^{\mathrm{+}}\text{-(in > out)}$ gradient conditions only with a physiological pH optimum between 7.0 and 7.5. It was also found that a pH-gradient (acidic outside) could stimulate both the Na⁺-gradient and the Na⁺ + K⁺-gradient-dependent transport of l-glutamic acid. However, pH- or K⁺-gradient alone were ineffective in stimulating uptake above simple diffusion level. Finally, it was found that increased rates of efflux were always observed with an acidic pH outside, whatever the conditions inside the vesicles. From these results, we propose a channel-type mechanism of l-glutamic acid transport in which Na⁺ and K⁺ effects are modulated by the surrounding pH. The model proposes a carrier with high or low affinity for Na⁺ in the protonated or unprotonated forms, respectively. We also propose that K⁺ binding occurs only to the unprotonated carrier and allows its fast recycling as compared to the free form of the carrier. Such a model would be maximally active and effective in the intestine in the in vivo physiological situations.     

Inhibition of human intestinal brush border membrane vesicle Na+-dependent phosphate uptake by phosphophloretin derivatives

Hyperphosphatemia and II(o) hyperparathyroidism are common and severe complications of chronic renal failure. Reduced dietary phosphorus has been shown to be an effective treatment in reducing serum phosphate and serum PTH. 2(')-Phosphophloretin inhibited small intestine apical membrane Na(+)/phosphate cotransport and reduced serum phosphate in adult rats. 2(')-PP and phosphoesters of phloretin were tested for inhibition of human small intestine brush border membrane alkaline phosphatase activity and for inhibition of Na(+)-dependent phosphate uptake. The IC(50)'s for inhibition of alkaline phosphatase suggested an order of inhibitory potency of 4-PP > phloretin > 4(')-PP > 2(')-PP. Inhibition of Na(+)-dependent phosphate uptake followed the sequence 2(')-PPz.Gt;4(')-PP > 4-PP > phloretin. These results are consistent with 2(')-PP being a specific inhibitor of human intestinal brush border membrane Na(+)/phosphate cotransport.     

Characteristics of glutamic acid transport by rabbit intestinal brush-border membrane vesicles. Effects of Na+-, K+- and H+-gradients

In the presence of a Na+-gradient (out greater than in), L-glutamic acid and L-and D-aspartic acids were equally well concentrated inside the vesicles, while no transport above simple diffusion levels was seen by replacement of Na+ by K+. Equilibrium uptake values were found inversely proportional to the medium osmolarity, thus demonstrating uptake into an osmotically sensitive intravesicular space. The extrapolation of these lines to infinite medium osmolarity (zero space) showed only a small binding component in acidic amino-acid transport. When the same experiment was performed at saturating substrate concentrations, linear relationships extrapolating through the origin but showing smaller slope values were recorded, thus indicating that the binding component could be more important than suspected above. However, binding to the membrane was neglected in our studies as it was absent from initial rate measurements. Na+-dependent uphill transport of L-glutamic acid was stimulated by K+ present on the intravesicular side only but maximal stimulation was recorded under conditions of an outward K+-gradient (in greater than out). Quantitative and qualitative differences in the K+ effect were noted between pH 6.0 and 8.0. Initial uptake rates showed pH dependency in Na+-(out greater than in) + K+-(in greater than out) gradient conditions only with a physiological pH optimum between 7.0 and 7.5. It was also found that a pH-gradient (acidic outside) could stimulate both the Na+-gradient and the Na+ + K+-gradient-dependent transport of L-glutamic acid. However, pH- or K+-gradient alone were ineffective in stimulating uptake above simple diffusion level. Finally, it was found that increased rates of efflux were always observed with an acidic pH outside, whatever the conditions inside the vesicles. From these results, we propose a channel-type mechanism of L-glutamic acid transport in which Na+ and K+ effects are modulated by the surrounding pH. The model proposes a carrier with high or low affinity for Na+ in the protonated or unprotonated forms, respectively. We also propose that K+ binding occurs only to the unprotonated carrier and allows its fast recycling as compared to the free form of the carrier. Such a model would be maximally active and effective in the intestine in the in vivo physiological situations.     

The mechanism of decreased Na+-dependent D-glucose transport in brush-border membrane vesicles from rabbit kidneys with experimental Fanconi syndrome

In our previous paper (Yanase, M. et al. (1983) Biochim. Biophys. Acta 733, 95-101) we reported that the Na+-dependent D-glucose uptake into brush-border membrane vesicles is decreased in rabbits with experimental Fanconi syndrome (induced by anhydro-4-epitetracycline). In the present paper we investigate the mechanism underlying this decrease. D-Glucose is taken up into the osmotically active space in anhydro-4-epitetracycline-treated brush-border membrane vesicles and exhibits the same distribution volume and the same degree of nonspecific binding and trapping as in control brush-border membrane vesicles. The passive permeability properties of control and anhydro-4-epitetracycline-treated brush-border membrane vesicles are shown to be the same as measured by the time-dependence of L-glucose efflux from brush-border membrane vesicles. D-Glucose flux was measured by the equilibrium exchange procedure at constant external and internal Na+ concentrations and zero potential. Kinetic analyses of Na+-dependent D-glucose flux indicate that Vmax in anhydro-4-epitetracycline-treated brush-border membrane vesicles (79.3 +/- 7.6 nmol/min per mg protein) is significantly smaller than in control brush-border membrane vesicles (141.3 +/- 9.9 nmol/min per mg protein), while the Km values in the two cases are not different from each other (22.3 +/- 0.9 and 27.4 +/- 1.8 mM, respectively). These results suggest that Na+-dependent D-glucose carriers per se are affected by anhydro-4-epitetracycline, and that this disorder is an important underlying mechanism in the decreased Na+-dependent D-glucose uptake into anhydro-4-epitetracycline-treated brush-border membrane vesicles.