Short-term studies of taurocholate uptake by ileal brush border membrane vesicles: Anion effects

Experiments allowing Na⁺-dependent short-term uptake measurements by ileal brush border vesicles were described. Glucose uptake was compared with taurocholate uptake in the presence of NaCl, NaSCN and Na₂SO₄. In contrast to the observation made with glucose, taurocholate transport was the same for the three electrolytes, indicating electroneutral taurocholate transport.     

Taurocholate uptake by membrane vesicles prepared from ileal brush borders

Taurocholate uptake by vesicles prepared from brush borders obtained from the small intestines of guinea pigs was studied. Vesicles obtained from the brush borders of ileums demonstrated an enhanced initial uptake in those incubations where a sodium ion gradient (extravesicular sodium concentration greater than intravesicular) was present at the outset. With the dissipation of this sodium gradient the intravesicular concentration of taurocholate declined. This overshoot phenomenon was absent in parallel incubations of vesicles made from jejunal tissue. When the sodium chloride was replaced by isosmotic amounts of mannitol no overshoot was observed in incubations of ileal vesicles until subsequent addition of sodium chloride to these incubations. These observations are in accord with the idea that those subcellular structural elements operating in the ileal bile salt transport system are associated with the brush border membranes of the ileal mucosal cells.     

Glycodeoxycholate transport in brush border membrane vesicles isolated from rat jejunum and ileum

The transport of the bile salt, glycodeoxycholate, was studied in vesicles derived from rat jejunal and ileal brush border membranes using a rapid filtration technique. The uptake was osmotically sensitive, linearly related to membrane protein and resembled d-glucose transport. In ileal, but not jejunal, vesicles glycodeoxycholate uptake showed a transient vesicle/medium ratio greater than 1 in the presence of an initial sodium gradient. The differences between glycodeoxycholate uptake in the presence and absence of a Na⁺ gradient yielded a saturable transport component. Kinetic analysis revealed a $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value similar to that described previously in everted whole intestinal segments and epithelial cells isolated from the ileum. These findings support the existence of a transport system in the brush border membrane that: (1) reflects kinetics and characteristics of bile salt transport in intact intestinal preparations, and (2) catalyzes the co-transport of Na⁺ and bile salt across the ileal membrane in a manner analogous to d-glucose transport.     

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     

Glycodeoxycholate transport in brush border membrane vesicles isolated from rat jejunum and ileum.

The transport of the bile salt, glycodeoxycholate, was studied in vesicles derived from rat jejunal and ileal brush border membranes using a rapid filtration technique. The uptake was osmotically sensitive, linearly related to membrane protein and resembled D-glucose transport. In ileal, but not jejunal, vesicles glycodeoxycholate uptake showed a transient vesicle/medium ratio greater than 1 in the presence of an initial sodium gradient. The differences between glycodeoxycholate uptake in the presence and absence of a Na+ gradient yielded a saturable transport component. Kinetic analysis revealed a Km value similar to that described previously in everted whole intestinal segments and epithelial cells isolated from the ileum. These findings support the existence of a transport system in the brush border membrane that: (1) reflects kinetics and characteristics of bile salt transport in intact intestinal preparations, and (2) catalyzes the co-transport of Na+ and bile salt across the ileal membrane in a manner analogous to D-glucose transport.     

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 (Na _out ⁺ >Na _in ⁺ ). 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.     

Transport of l-arginine in brush border vesicles derived from rabbit kidney cortex

The uptake of l-arginine by brush border vesicles from rabbit kidney cortex was investigated at 37 °C and pH 7.5. The initial rate of uptake (15 s) was twice as fast in a highly purified brush border as in brush border contaminated by basal-lateral plasma membranes. The initial uptake in a mannitol medium can be best described as the sum of transfer by two systems with K_m values of 0.07 and 3.5 mm and V_max values of 1.5 and 8 nmol/mg protein × 15 s, respectively. For the inhibitors of l-[¹⁴C]arginine, uptake (15 s at two substrate concentrations of 0.1 and 2.5 mm in a mannitol medium) the following sequence of inhibitory strength was established: l-arginine, l-ornithine, l-cystine, l-lysine, d-arginine, and NaCl. When a vesicular membrane potential was induced transiently by a jump of the pH in the incubation medium from 5.9 to 7.5 or by an outward movement of K⁺ in the presence of gramicidin D, an overshoot of l-arginine uptake was observed. Initial uptake of l-arginine was slightly faster in the presence of a Na⁺ gradient (outside to inside) than under a K⁺ gradient. Both ion gradients reduced uptake as compared to the uptake in a mannitol medium. Uptake was also studied after the membrane potential was minimized by equilibrating the vesicles in a NaCl or KC1 medium in the presence of gramicidin D. Under these conditions, l-arginine uptake in the first 30 s was faster in the NaCl than in the KCl medium. These experiments indicate, beside a major ion-independent l-arginine transport, the presence of a transport stimulated by Na⁺ in isolated brush border vesicles.     

Phenylalanine uptake in isolated renal brush border vesicles

The uptake of l-phenylalanine into brush border microvilli vesicles and basolateral plasma membrane vesicles isolated from rat kidney cortex by differential centrifugation and free flow electrophoresis was investigated using filtration techniques.Brush border microvilli but not basolateral plasma membrane vesicles take up l-phenylalanine by an Na⁺-dependent, saturable transport system. The apparent affinity of the transport system for l-phenylalanine is 6.1 mM at 100 mM Na⁺ and for Na⁺ 13 mM at 1 mM l-phenylalanine. Reduction of the Na⁺ concentration reduces the apparent affinity of the transport system for l-phenylalanine but does not alter the maximum velocity.In the presence of an electrochemical potential difference for Na⁺ across the membrane (η_Na0 >η_Na1) the brush border microvilli accumulate transiently l-phenylalanine over the concentration in the incubation medium (overshoot phenomenon). This overshoot and the initial rate of uptake are markedly increased when the intravesicular space is rendered electrically more negative by membrane diffusion potentials induced by the use of highly permeant anions, of valinomycin in the presence of an outwardly directed K⁺ gradient and of carbonyl cyanide p-trifluoromethoxyphenylhydrazone in the presence of an outward-directed proton gradient.These results indicate that the entry of l-phenylalanine across the brush border membrane into the proximal tubular epithelial cells involves cotransport with Na⁺ and is dependent on the concentration difference of the amino acid, on the concentration difference of Na⁺ and on the electrical potential difference. The exit of l-phenylalanine across the basolateral plasma membranes is Na⁺-independent and probably involves facilitated diffusion.     

Taurine transport by brush border membrane vesicles isolated from the flounder kidney

Summary Taurine transport was investigated in brush border membrane vesicles isolated from renal tubules of the winter flounder (Pseudopleuronectes americanus). Taurine uptake by the vesicles was greater in the presence of NaCl as compared to uptake in KCl. The Na⁺-dependent taurine transport was electrogenic and demonstrated tracer replacement and inhibition by -alanine and HgCl₂, indicating the presence of Na⁺-dependent, carrier-mediated taurine transport. In contrast to Na⁺-dependent taurine transport across the basolateral membrane, there was not a specific Cl^– dependency for transport in the brush border membrane. No evidence was obtained for Na⁺-independent carrier-mediated taurine transport. The possible involvement of the brush border Na⁺-dependent transport system in the net secretion of taurine from blood to tubular lumen in vivo (Schrock et al. 1982) is discussed.     

Effect of antiserum to a 99 kDa polypeptide on the uptake of taurocholic acid by rat ileal brush border membrane vesicles

A 99 kDa polypeptide in rat ileal brush border membrane (BBM), regarded as a component of the active bile acid transport system on account of photoaffinity labeling, has been purified by affinity chromatography and preparative gel electrophoresis and utilized as an immunogen for raising polyclonal antibody. Immune serum, but not preimmune serum, specifically recognized a single band of 99 kDa protein on immunoblots of ileal and renal BBM. In contrast, no reactivity was observed with proteins in jejunal BBM. This polyclonal antibody, compared with preimmune serum and anticytosolic bile acid binding protein (14 kDa) serum, significantly inhibited the Na+ dependent uptake of [3H] taurocholate by BBM vesicles (p less than 0.01). [14C] D-glucose uptake by BBM vesicles was not influenced by the immune serum (p less than 0.01). Thus, these studies provide further support for the specific role of a 99 kDa protein in ileal BBM bile acid transport.     

Bile acid uptake by isolated intestinal mucosa cells of guinea pig

Isolated intestinal mucosa cells of the guinea pig were employed to study intestinal transport of bile acids. Chenodeoxycholate and lithocholate were rapidly taken up into jejunal and ileal cells by diffusion. Taurocholate and cholate however showed only a minor diffusion rate and were preferentially taken up by the ileal bile acid carrier. This uptake was saturable with an apparent K_m of 231 μM and V of 7 nmol/mg protein per min for taurocholate; this bile acid was accumulated 90-fold. Its uptake was strongly inhibited by antimycin A, FCCP, ouabain or Na⁺-deficiency in the medium. Sugars or amino acids did not interfere with uptake. Experimental conditions were optimized with regard to incubation medium, cell amount, cell age and length of preincubation. It is concluded that ileal cells of the guinea pig are superior to other experimental models for characterizing the ileal bile acid carrier, because they allow us to determine initial rates of uptake and have a very efficient energetic coupling.     

Na+-independent l-arginine transport in rabbit renal brush border membrane vesicles

Na⁺-independent l-arginine uptake was studied in rabbit renal brush border membrane vesicles. The finding that steady-state uptake of l-arginine decreased with increasing extravesicular osmolality and the demonstration of accelerative exchange diffusion after preincubation of vesicles with l-arginine, but not d-arginine, indicated that the uptake of l-arginine in brush border vesicles was reflective of carrier-mediated transport into an intravesicular space. Accelerative exchange diffusion of l-arginine was demonstrated in vesicles preincubated with l-lysine and l-ornithine, but not l-alanine or l-proline, suggesting the presence of a dibasic amino acid transporter in the renal brush border membrane. Partial saturation of initial rates of l-arginine transport was found with extravesicular [arginine] varied from 0.005 to 1.0 mM. l-Arginine uptake was inhibited by extravesicular dibasic amino acids unlike the Na⁺-independent uptake of l-alanine, l-glutamate, glycine or l-proline in the presence of extravesicular amino acids of similar structure. l-Arginine uptake was increased by the imposition of an H⁺ gradient (intravesicular pH<extravesicular pH) and H⁺ gradient stimulated uptake was further increased by FCCP. These findings demonstrate membrane-potential-sensitive, Na⁺-independent transport of l-arginine in brush border membrane vesicles which differs from Na⁺-independent uptake of neutral and acidic amino acids. Na⁺-independent dibasic amino acid transport in membrane vesicles is likely reflective of Na⁺-independent transport of dibasic amino acids across the renal brush border membrane.     

Transport of β-alanine in renal brush border membrane vesicles

The findings that the equilibrium uptake of β-alanine decreased with increasing medium osmolarity and preincubation with β-alanine increased uptake of the amino acid indicate that the uptake of β-alanine by rabbit renal brush border membranes represents transport into membrane vesicles. A Na⁺ electrochemical gradient (extravesicular > intravesicular) stimulated the initial rate of β-alanine uptake about three times and effected a transient accumulation of the amino acid twice the equilibrium value. Stimulation of the uptake was specific for Na⁺. Gramicidin abolished the overshoot, presumably by dissipating the gradient by accelerating the electrogenic entrance of Na⁺ into the vesicle via a pathway not coupled to uptake of β-alanine. In K⁺-loaded vesicle, valinomycin enhanced the Na⁺ gradient-dependent uptake of β-alanine. These findings indicate that the Na⁺ gradient-dependent transport of β-alanine is an electrogenic process and suggest that the membrane potential is a determinant of β-analine transport. Uptake of β-aniline, at a given concentration, reflected the sum of contributions from Na⁺ gradient-dependent and -independent transport systems. The dependent system saturated at 100 μM. The independent system did not saturate. At physiological concentrations the rate of the Na⁺ gradient-dependent uptake was four times that in the absence of the gradient. The Na⁺ gradient-dependent rate of β-alanine uptake was strongly inhibited by taurine, suggesting that β-amino acids have a common transport system, α-Amino acids, i.e. l-arginine, l-glutamate, l-proline, and glycine, representing previously reported specific α-amino acid transport systems in the brush border membrane, did not inhibit the uptake of β-alanine. These findings indicate that the brush border membrane has a distinct transport system for β-amino acids.     

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.     

Regulation of canine renal vesicle Pi transport by growth hormone and parathyroid hormone

Renal phosphate (P_i) reabsorption is increased by growth hormone (GH) and decreased by parathyroid hormone (PTH). Na⁺-stimulated P_i transport across the brush border membrane of the proximal tubule is the initial step in the process of P_i reabsorption. To determine whether changes in P_i reabsorption induced by GH or PTH are accompanied by changes in brush border membrane Na⁺-gradient-stimulated P_i transport, we examined the effect of in vivo GH and PTH administration and thyroparathyroidectomy on P_i transport by isolated brush border membrane vesicles prepared from canine kidney. In experiments in which the effect of PTH administration was examined, the same animal provided the control kidney (before PTH administration) and the experimental kidney (after PTH administration). The Na⁺-gradient P_i overshoot in vesicles isolated from normal, GH-treated and thyroparathyroidectomized dogs was increased after in vivo PTH administration. GH administration and thyroparathyroidectomy increased the height of the overshoot compared to normal. PTH administration decreased the apparent $\text{V}$ value by 44% in vesicles from normal animals. The apparent $\text{V}$ value was increased, compared to normal, by GH (34%) and thyroparathyroidectomy (57%). PTH administration decreased the apparent $\text{V}$ in both the latter groups. GH administration to thyroparathyroidectomized dogs further increased the apparent $\text{V}$. Changes in the apparent $\text{V}$ paralleled changes in P_i reabsorption in vivo induced by experimental manipulations. We conclude that changes in renal P_i reabsorption induced by GH were like those induced by PTH, accompanied by changes in the Na⁺-stimulated P_i transport system in the renal brush border membrane, and that the effect of PTH on vesicular P_i transport in GH-treated dogs did not differ from the effect on vesicles from normal animals.     

Carrier-mediated transport of glycyl-L-proline in renal brush border vesicles

Renal brush border vesicles prepared from rabbit were shown to transport intact glycyl-L-proline into the intravesicular space by a Na⁺-independent, carrier-mediated process. The kinetics of hydrolysis of glycyl-L-proline by the vesicles showed that this dipeptide was extremely resistant to hydrolysis. The intravesicular concentration of glycyl-L-proline was the same in both NaCl and KCl media. Dipeptide uptake into the vesicles appeared to be via carrier-mediated transport down a concentration gradient.     

Renal palmitate transport: possible sites for interaction with a plasma membrane fatty acid-binding protein

Summary In previous studies from this laboratory [14], a mediated transport system for long chain fatty acids was observed in rat renal basolateral membrane vesicles. Transport was measured in the absence of albumin and indicated the presence of a Na⁺ independent anion exchange mechanism. The present experiments were done to characterize renal transport of fatty acids derived from fatty acid-albumin complexes. ³H-palmitate uptake by brush border (BBMV) and basolateral membrane vesicles (BLMV) isolated from rat renal cortex was determined using a rapid filtration technique. All incubation media contained 100 µM ³H-palmitate complexed to 100 µM bovine serum albumin. Up to 65% of initially bound fatty acid-albumin complexes were displaceable by washing with solution containing 0.1% albumin. Total palmitate uptake was measured as the remaining non-displaceable radioactivity. In BBMV in low ionic strength (300 mM mannitol) or ionic buffers (100 mM mannitol + 100 mM NaCl or KCl), total palmitate uptake at 15 sec did not differ from equilibrium (60 min) values of 10–11 nmoles/mg protein. Uptake was primarily due to binding. A similar pattern was seen with BLMV in 300 mM mannitol buffer: In BLMV in 100 mM NaCl or KCl buffers, equilibrium uptake was 10-fold lower than at 15 sec. This suggests binding followed by cation-dependent translocation. If a putative FABP_PM is involved in transport only, its presence should be confined to BLMV.     

Glycine transport by intestinal brush border vesicles of the amphibian Discoglossus pictus.

1. In order to determine the different components of glycine uptake by the intestine of the frog, Discoglossus pictus, we have used brush border membrane vesicles isolated by a classical precipitation technique. 2. Enzymatic tests showed that a good purification was obtained. The concentration ratio of alkaline phosphatase was 14.8. 3. Glycine entry in vesicles as a function of time, in presence or absence of sodium, indicated an overshoot which decreased when incubation time was prolonged. The overshoot was dependent on the presence of sodium. 4. The nature of the anion associated to sodium had little effect on glycine uptake. Nevertheless, chloride and thiocyanate appeared more efficient than glutarate. 5. The effect of transmembrane potential was studied by using valinomycin associated with a potassium gradient. The addition of this substance stimulated glycine transport by 43%. 6. The transport at different glycine concentrations showed two components: one non-saturable with weak affinity and the other saturable with strong affinity (Kt = 0.338 mM). 7. In conclusion, glycine transport by the brush border of D. pictus intestine presents a saturable component depending on sodium and on transmembrane electrical potential.     

Na+-dependent transport of glycine in renal brush border membrane vesicles: Evidence for a single specific transport system

The uptake of glycine in rabbit renal brush border membrane vesicles was shown to consist of glycine transport into an intravesicular space. An Na⁺ electrochemical gradient (extravesicular>intravesicular) stimulated the initial rate of glycine uptake and effected a transient accumulation of intravesicular glycine above the steady-state value. This stimulation could not be induced by the imposition of a K⁺, Li⁺ or choline⁺ gradient and was enhanced as extravesicular Na⁺ was increased from 10 mM to 100 mM. Dissipation of the Na⁺ gradient by the ionophore gramicidin D resulted in diminished Na⁺-stimulated glycine uptake. Na⁺-stimulated uptake of glycine was electrogenic. Substrate-velocity analysis of Na⁺-dependent glycine uptake over the range of amino acid concentrations from 25 μM to 10 mM demonstrated a single saturable transport system with apparent K_m = 996 μM and V_max = 348 pmol glycine/mg protein per min. Inhibition observed when the Na⁺-dependent uptake of 25 μM glycine was inhibited by 5 mM extravesicular test amino acid segregated dibasic amino acids, which did not inhibit glycine uptake, from all other amino acid groups. The amino acids d-alanine, d-glutamic acid, and d-proline inhibited similarly to their l counterparts. Accelerative exchange of extravesicular [³H]glycine was demonstrated when brush border vesicles were preloaded with glycine, but not when they were preloaded with l-alanine, l-glutamic acid, or with l-proline. It is concluded that a single transport system exists at the level of the rabbit renal brush border membrane that functions to reabsorb glycine independently from other groups of amino acids.     

Decreased Na+-gradient-dependent d-glucose transport in brush-border membrane vesicles from rabbits with experimental fanconi syndrome

The effect of anhydro-4-epitetracycline on sodium gradient-dependent d-glucose transport of rabbit renal brush-border membrane vesicles was studied. The purity of isolated brush-border membrane vesicles as judged by enzyme activities was not different between normal control and anhydro-4-epitetracycline-administered rabbits. There was no difference in estimate of intravesicular volume, either. When NaCl was used for sodium gradient, the overshoot of d-glucose uptake into brush-border membrane vesicles isolated from anhydro-4-epitetracycline-treated rabbits was significantly smaller than that of normal control rabbits. In the cases of NaSCN or Na₂SO₄, the former was also smaller than the latter, but not significantly so. To avoid the possible effect of membrane potential on d-glucose uptake, the voltage-clamp method was applied. Even in the voltage-clamped condition, the overshoot of d-glucose uptake into vesicles from anhydro-4-epitetracycline-treated rabbits was decreased compared to that of normal rabbits. In vitro incubation of brush-border membrane vesicles with 20 mM anhydro-4-epitetracycline caused no alteration in sodium gradient-dependent d-glucose uptake. Our results demonstrate that there exists a disorder in sodium gradient-dependent d-glucose uptake of renal brush-border membrane in anhydro-4-epitetracycline-treated rabbits, and suggest that this disorder is one of the underlying mechanisms of experimental Fanconi syndrome.