Role of alkaline phosphatase in phosphate uptake into brush border membrane vesicles from human intestinal mucosa

In order to elucidate the physiological function of intestinal alkaline phosphatase, the characteristics of human intestinal alkaline phosphatase bound to brush border membrane vesicles were compared under optimal and physiological pHs. The Km value of this enzyme towards p-nitrophenylphosphate at the physiological pH was lower than that at the optimal pH. At the physiological pH, phosphate, arsenate and vanadate competitively inhibited the alkaline phosphatase activity, as they did at optimal pH, and the K1 values of these inhibitors at the physiological pH were also lower than those at the optimal pH. The effects of various inhibitors and antibody to human intestinal alkaline phosphatase on phosphate uptake into brush border membrane vesicles were investigated. The results indicated that phosphate uptake was affected by various inhibitors and the antibody to human intestinal alkaline phosphatase, but L-homoarginine, levamisole, and ouabain had no effect. From the above findings, it is strongly suggested that human intestinal alkaline phosphatase may function as a phosphate binding protein at low phosphate concentrations under physiological conditions.     

Calcium and glucose uptake in rat small intestinal brush-border membrane vesicles. Modulation by exogenous hypercortisolism and 1,25-dihydroxyvitamin D-3

The effect of exogenous hypercortisolism and 1,25-dihydroxyvitamin D-3 on small-intestinal calcium and glucose transport in the rat was studied at the level of brush-border membrane vesicles generated from isolated villous cells by a freeze-thaw procedure. At 5 X 10(-5) M extravesicular calcium, initial uptake rates in vesicles prepared from triamcinolone-treated adult rats were decreased by 30% after 5 days. Since calcium ionophore A23187 virtually abolished the difference in calcium uptake, triamcinolone appeared to affect calcium channel density or activity rather than intravesicular binding capacity. Kinetic analysis showed that a decrease in Vmax of a saturable calcium transport system could entirely account for the diminished rate of vesicular calcium uptake. Calcium transport rates could be partially restored by in vivo administration of 1,25-dihydroxyvitamin D-3 at a dosage which did not affect vesicular calcium uptake in control animals. Conversely, sodium-driven glucose accumulation in brush-border vesicles from triamcinolone-treated rats was stimulated by 50-70% after 36 h and appeared insensitive to vitamin D. A specific triamcinolone action on the glucose carrier itself rather than on the driving force of the sodium gradient was indicated by (i) a similar stimulation of glucose transport under equilibrium exchange conditions and (ii) an opposite effect of triamcinolone on sodium-driven alanine transport. The triamcinolone-induced changes in calcium and glucose uptake were not accompanied by a gross alteration of membrane integrity in vitro or by major alterations in vesicular protein composition, intravesicular glucose space and sucrase or alkaline phosphatase activity. The modification of vesicular transport properties is discussed in relation to the vitamin D-antagonized inhibition of intestinal calcium uptake and the stimulation of glucose absorption in response to supraphysiologic amounts of glucocorticoids observed in intact epithelium.     

Hibernation enhancesd-glucose uptake by intestinal brush border membrane vesicles in ground squirrels

The ability to actively transport nutrients is maintained in intestinal tissues of hibernating ground squirrels compared with their active counterparts, and shows apparent upregulation in hibernators when transport rates are normalized to tissue mass. To identify the mechanisms responsible for the preservation of transport function during the extended fast of hibernation, we studiedd-glucose uptake into jejunal brush border membrane vesicles prepared from active and hibernating 13-lined ground squirrels. Hibernators were without food and showing regular bouts of torpor for at least 6 weeks before sacrifice. Electron micrographs indicated similar microvillus heights of jejunal enterocytes in the two activity states, whereas microvillus density was slightly greater in the hibernators. Glucose uptake into brush border membrane vesicles was inversely related to medium osmolarity, indicating negligible binding of substrate to brush border membrane vesicles surfaces, and intravesicular spaces were similar in hibernating and active squirrels. Glucose uptake showed strong Na⁺ dependency in both groups, with equivalent overshoot values in the presence of Na⁺. Kinetic analysis revealed a significant increase in the maximal velocity of transport (J _max) in hibernators (55.9±5.6 nmol·min^-1·mg^-1) compared with active squirrels (36.7±5.1 nmol·min^-1·mg^-1,P<0.05), with no change inK _m. Thus, the structure and absorptive capacity of the intestinal brush border persists in fasted hibernators, and the increase inJ _max for glucose uptake during hibernation likely contributes to the enhanced Na⁺-dependent glucose absorption previously observed at the tissue level.Abbreviations BBM brush border membrane(s) - BBMV brush border membranes vesicles - SGLT1 Na⁺-glucose transporter - 3-OMG 3-orthomethylglucose - J _max maximal velocity of transport - K _m transporter affinity for substrate - T _b body temperature     

Calcium accumulation by chick intestinal mitochondria: Regulation by vitamin D-3 and 1,25-dihydroxyvitamin D-3

We have evaluated the effect of vitamin D-3 and its metabolite 1,25-dihydroxyvitamin D-3 on Ca²⁺ accumulation by chick intestinal mitochondria. Ca²⁺ accumulation appears to occur in two phases: an early, transient accumulation into an Na⁺-labile pool followed by an ATP-dependent accumulation into an Na⁺-resistant pool. Ca²⁺ accumulation is extensive at free Ca²⁺ concentrations greater than 3 · 10^?6 M in the presence of ATP. Ruthenium red and dinitrophenol block Ca²⁺ accumulation, but atractyloside does not. Oligomycin blocks ATP-supported accumulation completely with a partial inhibition of ATP and malate-supported accumulation. Little difference could be found in mitochondrial preparations from vitamin D-deficient chicks compared to those from vitamin D-3 (or 1,25(OH)₂D-3)-supplemented chicks with respect to respiratory control, oxygen consumption, efficiency of oxidative phosphorylation, affinity for Ca²⁺, or the rate and extent of ATP-supported Ca²⁺ accumulation. Intestinal cytosol stimulated Ca²⁺ accumulation, but this was not specific with respect to vitamin D status or tissue of origin, nor was it duplicated by chick intestinal Ca²⁺-binding protein. 30 ng/ml 1,25(OH)₂D-3 stimulated Ca²⁺ accumulation directly, regardless of the presence of intestinal cytosol. Other vitamin D metabolites were less potent: 25-hydroxyvitamin D-3 > 24,25-dihydroxyvitamin D-3 = vitamin D-3. Since increasing the free Ca²⁺ concentration from 3 · 10^?6 to 1 · 10^?5 M increased Ca²⁺ accumulation approx. 50-fold, whereas direct stimulation by 1,25(OH)₂D-3 in vitro increased Ca²⁺ accumulation less than 2-fold, we conclude that 1,25(OH)₂D-3 influences mitochondrial accumulation of Ca²⁺ in vivo primarily by altering cytosol concentrations of free Ca²⁺.     

Calcium accumulation by chick intestinal mitochondria. Regulation by vitamin D-3 and 1,25-dihydroxyvitamin D-3

We have the evaluated the effect of vitamin D-3 and its metabolite 1,25-dihydroxyvitamin D-3 on Ca2+ accumulation by chick intestinal mitochondria. Ca2+ accumulation appears to occur in two phases: an early, transient accumulation into an Na+-labile pool followed by an ATP-dependent accumulation into an Na+-resistant pool. Ca2+ accumulation is extensive at free Ca2+ concentrations greater than 3 . 10(-6) M in the presence of ATP. Ruthenium red and dinitrophenol block Ca2+ accumulation, but atractyloside does not. Oligomycin blocks ATP-supported accumulation completely with a partial inhibition of ATP and malate-supported accumulation. Little difference could be found in mitochondrial preparations from vitamin D-deficient chicks compared to those from vitamin D-3 (or 1,25(OH)2D-3)-supplemented chicks with respect to respiratory control, oxygen consumption, efficiency of oxidative phosphorylation, affinity for Ca2+, or the rate and extent of ATP-supported Ca2+ accumulation. Intestinal cytosol stimulated Ca2+ accumulation, but this was not specific with respect to vitamin D status or tissue of origin, nor was it duplicated by chick intestinal Ca2+-binding protein. 30 ng/ml 1,25(OH)2D-3 stimulated Ca2+ accumulation directly, regardless of the presence of intestinal cytosol. Other vitamin D metabolites were less potent: 25-hydroxyvitamin D-3 greater than 24,25-dihydroxyvitamin D-3 = vitamin D-3. Since increasing the free Ca2+ concentration from 3 . 10(-6) to 1 . 10(-5) M increased Ca2+ accumulation approx. 50-fold, whereas direct stimulation by 1,25(OH)2D-3 in vitro increased Ca2+ accumulation less than 2-fold, we conclude that 1,25(OH)2D-3 influences mitochondrial accumulation of Ca2+ in vivo primarily by altering cytosol concentrations of free Ca2+.     

Ion permeability of rabbit intestinal brush border membrane vesicles

Summary The ion permeability of rabbit jejunal brush border membrane vesicles was studied by measuring unidirectional fluxes with radioactive tracers and bi-ionic diffusion potentials with the potential-sensitive fluorescent dye, diS–C₃-(5). Tracer measurements provide estimates of the absolute magnitudes of permeability coefficients, while fluorescence measurements provide estimates of relative and absolute ion permeabilities. The magnitudes of the permeability coefficients for Na⁺, K⁺, Rb⁺, and Br^– were approximately 5 nanoliters/(mg protein × sec) or 10^–5 cm/sec as determined by radioactive tracer measurements. The apparent selectivity sequence, relative to Na⁺, as determined by bi-ionic potential measurements was: F^–, isetheionate, gluconate, choline (<0.1)+(1.0)–(1.5)=NO ₃ ^– (1.5)–(2.3)+(2.4)+(2.5)+(2.6)+(3.9) 4 ^– +(12)–(40). The origin of this selectivity sequence and its relationship to the ion permeability of the brush border membrane in the intact epithelium are discussed.     

Effect of papain treatment on dipeptide transport into rabbit intestinal brush border vesicles

Purified rabbit intestinal brush border membrane vesicles transport glycyl-L-proline into an osmotically responsive intravesicular space by a Na⁺- independent, carrier-mediated process. With short incubation, transport occurs mostly as the intact dipeptide, followed by hydrolysis. Pretreatment of the vesicles with papain results in a 60% reduction of L-alanine transport while glycyl-L-proline transport is stimulated by 40%. Papain treatment does not change the intravesicular volume, nor does it increase membrane permeability. Dipeptide transport into papain treated vesicles remains completely Na⁺- independent as it is in the control vesicles. Many dipeptides inhibit glycyl-L-proline transport into papain treated vesicles both in the presence and absence of a Na⁺ gradient.     

Effects of 1,25-dihydroxyvitamin D-3 on phospholipid metabolism in chick myoblasts

1,25-Dihydroxyvitamin D-3 has been shown to increase phosphatidylcholine and decrease phosphatidylethanolamine levels of myoblasts. Recent studies have suggested that the metabolite stimulates the methylation of phosphatidylethanolamine into phosphatidylcholine. In addition, the sterol increases the arachidonate content of phosphatidylcholine. Experiments were carried out to identify the steps of muscle cell lipid metabolism affected by 1,25-dihydroxyvitamin D-3. Primary cultures of chick embryo myoblasts pretreated with physiological concentrations of 1,25-dihydroxyvitamin D-3 were labelled with [14C]ethanolamine. The sterol increased the incorporation of precursor into dimethylphosphatidylethanolamine and phosphatidylcholine, whereas it decreases the labelling of phosphatidylethanolamine. Prior treatment with cycloheximide and actinomycin D blocked these changes. 1,25-Dihydroxyvitamin D-3 also stimulated the incorporation of [14C]ethanolamine into CDP-ethanolamine. In addition, the sterol increased the incorporation of [3H]arachidonic acid into the phosphatidylcholine fraction but did not affect the incorporation of [14C]palmitic acid. The incorporation of labelled fatty acids into diacylglycerol was not changed by the sterol, whereas it stimulated incorporation of both precursors into triacylglycerol. The data indicate that 1,25-dihydroxyvitamin D-3 enhances the synthesis of phosphatidylcholine through a stimulation of de novo synthesis and methylation of phosphatidylethanolamine via a nuclear mechanism. The sterol may also increase the polyunsaturated fatty acid content of phosphatidylcholine by means of an activation of its deacylation-reacylation cycle.     

Peptide transport in intestinal and renal brush border membrane vesicles

A longstanding question about the possible dependence of transmembrane peptide transport on sodium has now been resolved. Recent studies with purified intestinal brush border membrane vesicles have shown that peptide transport across this membrane is Na⁺-independent and occurs by a non-concentrative mechanism. Similar studies with renal brush border membrane vesicles have established for the first time the presence of a peptide transport system in mammalian kidney. The essential characteristics of peptide transport in these two tissues are the same. However, it still remains to be seen whether a new mechanism other than the Na⁺-gradient, hitherto unrecognized, is involved in energizing the active transport of peptides in vivo in mammalian intestine and kidney.     

Effect of temperature and pH on phosphate transport through brush border membrane vesicles in rats

The kinetics of sodium gradient dependent phosphate uptake by the renal brush border membrane vesicles of the rat have ben studied under various conditions of temperature and pH. From 7 to 30 degrees C the Lineweaver-Burk plots are linear, and the apparent Km progressively increases from 54 to 91 microM. Above 30 degrees C, the apparent Km continues to increase to reach 135 microM at 40 degrees C, but a break is observed in the Lineweaver-Burk plots at the substrate concentration of 300 microM. The existence of this break, confirmed by the Eadie-Hofstee plot supports the hypothesis of a dual mechanism of phosphate transport, one for low concentrations of substrate with a Km of 100 microM and the other for high concentrations with a Km of approximately 240 microM. When the two components of the Eadie-Hofstee plot are analyzed according to a nonlinear regression program, these two values of Km become 70 microM and 1.18 mM, respectively. The Vmax continuously increases with temperature. However, the Arrhenius plot (In Vmax vs. 1/TK) shows an abrupt discontinuity at 23 degrees C. pH experiments were performed at 35 degrees C. In the absence of a proton gradient, increasing the pH from 6.5 to 7.5 and 8.5 decreases the apparent Km from 341 to 167 and 94 microM, respectively. When only the divalent form of phosphate is considered as the substrate, the apparent Km does not vary anymore with the pH and remains around the mean value of 105 microM. The uniformity of the apparent Km for the total phosphate uptake, when only the divalent phosphate is considered as being the substrate, suggests that this divalent form is the only one which is transported. Whatever the substrate considered, total phosphate or divalent phosphate, the highest Vmax is obtained at pH 7.5 which probably approximates the optimum pH inside the vesicles for the phosphate uptake.     

Effects of long- and medium-chain triglycerides on amino acid uptake in rat intestinal brush border membrane vesicles

• 1.1. Uptake of l-leucine, l-phenylalanine, l-proline and l-lysine into brush border membrane vesicles from rats fed either a medium-chain triglyceride (MCT) or a long-chain triglyceride (LCT) diet was studied under conditions of the presence or absence of a Na⁺ gradient.
• 2.2. From the results of initial rate, Na⁺-dependent transport in LCT feeding were lower than in feeding MCT. The Na⁺-independent transport did not vary in either group except for l-lysine uptake.
• 3.3. For l-leucine, l-phenylalanine and l-proline in Na⁺ dependence, kinetic analysis revealed 4–6-fold smaller V_max values in LCT group than in MCT group. l-Lysine in Na⁺-independent transport was 10-fold lower in LCT group than in MCT group. The K_m values were not affected by feeding the LCT or MCT diet.
• 4.4. It is clear that amino acid transport is regulated by different types of dietary fat. We consider that the alteration of transport activity is attributable to the changes in number of membrane-bound transport carriers but not to their affinity.

     

Bile acid uptake and calcium flux in brush border membrane vesicles

Our laboratory has recently reported that intestinal bile acid malabsorption in cystic fibrosis (CF) is a primary mucosal cell defect. Others have suggested that elevated intracellular Ca⁺⁺ levels in other cell types in CF may represent a common primary dysfunction in Ca⁺⁺ efflux in these cells. We examined the possibility that intestinal bile acid absorption and Ca⁺⁺ efflux in mucosal cells may be linked physiologically. Brush border membrane vesicles (BBMV) prepared from guinea pig ileum served as the experimental model to test this hypothesis. Ca⁺⁺ (2.5×10^?3M) present in the incubation medium did not alter the uptake of taurocholic acid (TCA) by BBMV. Also, TCA uptake into BBMV preloaded with Ca⁺⁺ was not significantly different from that in BBMV not previously loaded with Ca⁺⁺. Furthermore, with TCA present in the incubation medium, Ca⁺⁺ efflux from preloaded BBMV was not altered. These data suggest that ileal TCA uptake, as measured by BBMV, is not dependent upon either intra- or extravesicular Ca⁺⁺. Also, Ca⁺⁺ efflux from BBMV is unaffected by TCA uptake. Although separate lines of evidence suggest that intestinal bile acid malabsorption and reduced plasma membrane Ca⁺⁺ flux are primary defects in CF, we conclude that in the normal intestine these functions are independent physiological processes.     

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.     

Na+-independent sugar uptake by rat intestinal and renal brush border and basolateral membrane vesicles

• 1.1. Uptake of [¹⁴C]-labelled d-glucose, l-arabinose and d-fructose by intestinal and renal brush border and basolateral membrane vesicles was studied in the absence of Na⁺ .
• 2.2. The Na⁺-independent d-glucose transport system in these membrane vesicles was saturable, sensitive to phloretin, stereospecific and accessible only to d-glucose and d-galactose.
• 3.3. Na⁺-independent l-arabinose transport was not saturable even when its concentration was raised to 300 mM and it was insensitive to phloretin.
• 4.4. Na⁺-independent d-fructose transport demonstrated saturation kinetics with only renal brush border membrane vesicles, but it was not inhibited by either phloretin or phlorizin.
• 5.5. These studies indicated that the Na⁺-independent carrier-mediated d-glucose/d-galactose transport system of intestinal and renal brush border and basolateral membranes is clearly not shared by other monosaccharides.

     

l-Arginine uptake into renal brush border membrane vesicles

The uphill uptake of l-arginine by renal brush border membrane vesicles was found to be energized by a Na⁺ gradient (extravesicular > intravesicular) in the presence of a membrane potential (inside negative). The uptake was specific for Na⁺. Either a K⁺-diffusion potential, generated by valinomycin, or a H⁺-diffusion potential, generated by the mitochondrial uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, provided the electrical driving force. The Na⁺ gradient-dependent l-arginine transport system was shared by specific basic amino acids and l-cystine, but not by d-arginine nor other classes of amino acids. The molecular structure of the basic amino acid recognized by the carrier was postulated.     

Riboflavin uptake by rat small intestinal brush border membrane vesicles: A dual mechanism involving specific membrane binding

The first step of riboflavin absorption was studied by determining the uptake of the vitamin by rat small intestinal brush border membrane vesicles. Vesicles were incubated at 25°C in the presence of [³H]-riboflavin at concentrations within the physiological intraluminal range for rat. The time course of [³H]-riboflavin uptake was unaffected by Na⁺ or K⁺ gradients. The 5 sec uptake rate plotted as a function of the initial concentration of [³H]-riboflavin in the medium (0.125 to 7.5 m) revealed the presence of a dual mechanism, with a saturable component (apparent kinetic constants: 0.12 m for K _m and 0.36 pmol · mg^-1 protein · 5 sec^-1 for J _max) prevailing at low concentrations (<2 m), and a nonsaturable component prevailing at higher concentrations. The presence of a carrier-mediated system for riboflavin was validated by counter-transport experiments. At equilibrium, uptake was almost completely accounted for by membrane binding, whereas at earlier times the transport component accounted for about 30% of total uptake. The plot of [³H]-riboflavin binding at equilibrium as a function of its concentration in the medium was quite similar to that of the 5 sec uptake rate in both intact and osmotically shocked vesicles and demonstrated the occurrence of a saturable component: binding constants were 0.07 (K _d) in m), 0.54 (B _max in pmol · mg^-1 protein), and 0.11 (K _d), 1.13 (B _max, respectively, indicating the existence of specific riboflavin binding sites. The specificity of riboflavin binding to the membrane was confirmed by preliminary studies with structural analogues. Specific binding could represent the first step of a specific riboflavin entry mechanism in enterocytes.This research was supported by grants from Italian MPI 60% (1989, 1992) and CNR n. 90, 02467 CT 04. We wish to express our gratitude to Prof. E. Perucca (Department of Internal Medicine, Clinical Pharmacology Unit, University of Pavia) for revising the English, and to Mrs. M. Agrati Greco and Mrs. P. Vai Gatti for secretarial assistance and excellent typing.     

Glycine uptake in brush border vesicles isolated from the rat intestinal cells

The uptake of glycine in osmotically active brush border membrane vesicles (obtained by the Mg++ precipitation method) has been studied and a partial characterization of its transport system has been established. The glycine uptake in these vesicles was stimulated by the presence of sodium and in the presence of an inwardly directed Na+ -gradient glycine was accumulated inside the vesicles. The effect of Na+ was specific; other monovalent cation as Li+, K+, Rb+ and choline were uneffective in the stimulation of glycine uptake, under the same experimental conditions. Preliminary experiments show an important role of some anions on the glycine uptake. A strong inhibition in the uptake rate was found when the measurements were carried out in the presence of sodium cyclamate, while in the presence of NaSCN the measured uptake values were similar to those observed in the presence of NaCl.     

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.