Methodological aspects of measuring amino acid uptake in studies with porcine jejunal brush border membrane vesicles

With L-glutamine, as a representative amino acid this study was undertaken to examine the effects of substrate concentrations on initial and equilibrium amino acid uptake and intravesicular volume determined with porcine jejunal brush border membrane vesicles prepared by Mg2+-aggregation and differential centrifugation. Transport measurements (24 degrees C) were conducted by the rapid filtration manual procedure. Glutamine uptake was shown to occur into an osmotically-active space ranging between 1.09-1.58 microl/mg protein with little non-specific membrane binding. At different concentrations (in parentheses), the duration of initial glutamine uptake in both Na+ gradient and Na+-free conditions was 10 s (0.01 mM), 15 s (0.17 mM), and 20 s (1.9 and 9.4 mM), respectively. Substrate concentrations affected the duration of initial uptake, with lower substrate concentrations giving shorter duration for initial amino acid uptake. At different substrate concentrations (in parentheses), the time required to reach equilibrium glutamine uptake was 5 min (0.01 mM), 10 min (0.17 mM), and 60 min (1.9 and 9.4 mM), respectively. Thus, substrate concentrations also affected the time required to reach equilibrium uptake. The higher the substrate concentration, the longer the incubation time needed to reach equilibrium amino acid uptake. At the glutamine concentrations of 0.01, 0.17, 1.9, and 9.4 mM, the average intravesicular volume was estimated to be 1.58+/-0.21, 1.09+/-0.28, 1.24+/-0.18, and 1.36+/-0.21 microl/mg protein, respectively. Substrate concentrations had no effect (p>0.05) on the intravesicular volume of membrane vesicles. In conclusion, in the experiments on amino acid transport kinetics measured with the rapid filtration manual procedure, the incubation time used for measuring the initial uptake rate should be determined from the time course experiments conducted at the lowest substrate concentration used, whereas the intravesicular volume can be obtained from equilibrium uptake measured at any substrate concentrations.     

pH-dependent heterogeneity of acidic amino acid transport in rabbit jejunal brush border membrane vesicles.

Initial rates of Na(+)-dependent L-glutamic and D-aspartic acid uptake were determined at various substrate concentrations using a fast sampling, rapid filtration apparatus, and the resulting data were analyzed by nonlinear computer fitting to various transport models. At pH 6.0, L-glutamic acid transport was best accounted for by the presence of both high (Km = 61 microM) and low (Km = 7.0 mM) affinity pathways, whereas D-aspartic acid transport was restricted to a single high affinity route (Km = 80 microM). Excess D-aspartic acid and L-phenylalanine served to isolate L-glutamic acid flux through the remaining low and high affinity systems, respectively. Inhibition studies of other amino acids and analogs allowed us to identify the high affinity pathway as the X-AG system and the low affinity one as the intestinal NBB system. The pH dependences of the high and low affinity pathways of L-glutamic acid transport also allowed us to establish some relationship between the NBB and the more classical ASC system. Finally, these studies also revealed a heterotropic activation of the intestinal X-AG transport system by all neutral amino acids but glycine through an apparent activation of Vmax.     

Kinetic analysis of L-glutamine transport into porcine jejunal enterocyte brush-border membrane vesicles

L-Glutamine transport into porcine jejunal enterocyte brush border membrane vesicles was studied. Uptake was mediated by a Na(+)-dependent and a Na(+)-independent pathway as well as by diffusion. The initial rates of glutamine uptake over a range of concentrations is both Na(+)-gradient and Na(+)-free conditions were analyzed and kinetic parameters were obtained. Na(+)-dependent glutamine transport had a K(m) of 0.77 +/- 0.16 mM and a Jmax of 70.7 +/- 5.8 pmol mg protein-1 s-1; Na(+)-independent glutamine transport had a K(m) of 3.55 +/- 0.78 mM and a Jmax of 55.1 +/- 6.6 pmol mg protein-1 s-1. The non-saturable component measured with HgCl2-poisoned brush border membrane vesicles in the Na(+)-free condition contained passive diffusion and non-specific membrane binding and was defined to be apparent glutamine diffusion and the glutamine permeability coefficient (Kdiff) was estimated to be Kdiff = 3.78 +/- 0.06 pmol 1 mg protein-1 mmol-1 s-1. Results of inhibition experiments showed that Na(+)-dependent glutamine uptake occurred primarily through the brush border system-B degree transporters, whereas Na(+)-independent glutamine uptake occurred via the system-L transporters. Furthermore, the kinetics of L-leucine and L-cysteine inhibition of L-glutamine uptake demonstrated that neutral amino acids sharing the same brush border transporters can effectively inhibit each other in their transport.     

Characterization of lipid intake into rabbit jejunal brush border membrane vesicles.

The absorption of lipids is generally accepted to be mediated by a process of passive diffusion, although some recent data have raised the possibility of a mediated component. Brush border membrane vesicles (BBMV) have been widely used to study nutrient transport, but have only recently been used to examine the uptake of lipids. Using a BBMV technique validated with studies of the uptake of D-glucose, we examined the uptake of linoleic acid into the jejunum of adult rabbits. The uptake of 100 microM linoleic acid was constant between 2 and 20 min, with no overshoot observed at earlier periods. Linoleic acid uptake was suppressed by 88% with 0.6 mM phloridzin and by 58% with 0.2 mM phloretin, but uptake of linoleic acid was unaffected by the absence of sodium, by the presence of a sodium gradient, or by varying the osmolarity of the buffer. Lysis of the BBMV incubated with linoleic acid by the addition of ice-cold deionized water did not alter the amount of linoleic acid associated with the BBMV. The linoleic acid concentration curve was linear up to 160 microM, when carried out under initial rate conditions and in the presence of 2 mM taurocholic acid. These results are compatible with the process of passive uptake of linoleic acid into BBMV of rabbit jejunum, but do not exclude the possible physiological importance of a membrane fatty acid binding protein.     

Activation of chloride conductance in pig jejunal brush border vesicles

Summary Requirements for the activation of Cl conductance have been investigated in pig jejunal brush border vesicles. The stability of ATP as a substrate for protein kinase activity, the stability of the phosphoprotein product of protein kinase action, and the choice of buffer system used for vesicle preparation were studied as variables which affected the outcome of in vitro activation attempts. Arsenate was selected as the most effective agent in protecting ATP from hydrolysis by the phosphatase activity in this vesicle system. Brush border vesicle protein appeared to prevent the accumulation of phosphoprotein in a cAMP-dependent protein kinase reaction, and vesicle protein only had phosphate acceptor activity when KF was added as a presumptive inhibitor of phosphoprotein phosphatase.A Cl conductance response to a potassium gradient and valinomycin was present in vesicles prepared in buffers containing tetramethylammonium. Cl^– conductance activity was not increased in this system by the addition of ATP, dibutyryl cyclic AMP, and cyclic AMP-dependent protein kinase.There was no Cl conductance response to a potassium gradient in vesicles buffered with imidazolium-acetate. Incorporation of ATP, AsO ₄ ^3– , and F^– into these nonconductive vesicles by homogenization, followed by addition of dibutyryl cAMP, produced substantial conductance activity. Maximal activation of Cl^– conductance was obtained with vesicles prepared in imidazolium-acetate buffering, using precautions to stabilize ATP and phosphoprotein prior to conductance measurements.     

Cholera toxin facilitates calcium transport in jejunal brush border vesicles

Cholera toxin is very well characterized in terms of the activation of adenylate cyclase. In some systems, however, this cyclase activation does not seem to account for all of the physiological responses to the toxin. On the premise that cholera toxin may also exert effects through other second messenger compounds we have studied the effect of cholera toxin on the rate of Ca2+ movement across the membrane of intestinal brush border vesicles. Increasing concentrations of cholera toxin progressively accelerated the passive uptake of Ca2+ into, and the efflux of Ca2+ from, an osmotically active space in brush border membrane vesicles. This effect of cholera toxin was saturable by excess Ca2+ and was relatively specific, as the toxin did not affect vesicle permeability to an uncharged polar solute. The toxin had two high affinity Ca2+ binding sites on the A subunit as measured by equilibrium dialysis. Ca2+ transport facilitated by cholera toxin was temperature dependent, required the holotoxin, and could be inhibited by preincubation of the toxin with excess free ganglioside GM1. This increased rate of Ca2+ influx caused by the in vitro addition of cholera toxin to brush border membrane vesicles may have physiological significance as it was comparable to rates observed with the Ca ionophore A23187. Similar effects occurring in vivo could permit cholera toxin to increase cytoplasmic Ca2+ concentrations and to produce accompanying second messenger effects.     

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.     

Ricinoleate and deoxycholate are calcium ionophores in jejunal brush border vesicles

Summary The intestinal secretagogues ricinoleate and deoxycholate have been tested for a capacity to form complexes with Ca²⁺ ions and to affect the passive equilibration of Ca²⁺ ions across the jejunal brush border membrane. Both of these agents formed butanol-soluble Ca²⁺ complexes in a model phase distribution system. They also promote the passive uptake and efflux of Ca²⁺ across brush border vesicles in a concentrationdependent manner. The levels of ricinoleate and deoxycholate that increase the rate of transvesicular Ca²⁺ movement are in the 100 to 300 m range. Concentrations as high as 1.0mm had no significant detergent effects in vesicles as measured by release of entrapped sorbitol. The kinetics of Ca²⁺ uptake and efflux are similar in brush border vesicles treated with A23187, ricinoleate, or deoxycholate. The influx rates observed in this study were high enough to cause the collapse of a Ca²⁺ gradient, which had been generated by Ca-Mg ATPase enzyme activity in the brush border membrane. Ricinoleate did not affect Ca-Mg ATPase activity at concentrations used in this study, but deoxycholate was inhibitory, indicating two potential modes for elevation of intracellular Ca²⁺ content by deoxycholate. When compared with the effects of the Ca²⁺ ionophore, A23187, it appears that both ricinoleate and deoxycholate could have significant intestinal secretory activity due to this Ca²⁺ ionophore property. It is also noteworthy that, at least in this model system, potential secretory effects are expressed at concentrations significantly below levels that have been associated with detergent effects or altered epithelial morphology.     

Ketone body transport in renal brush border membrane vesicles

Ketone body uptake by renal brush border vesicles has been investigated. Ketone bodies enter into the brush border vesicles by a carrier-mediated process. The uptake is dependent on an Na⁺ gradient ([Na⁺]outside > [Na⁺]inside) and is electroneutral. The uptake is transport into an osmotically active space and not a binding artifact as indicated by the effect of increasing the medium osmolarity. A pH gradient (alkaline inside) also stimulates the ketone body uptake. Acetoacetate and 3-hydroxybutyrate share the same carrier as demonstrated by the accelerated exchange diffusion and mutual inhibitory effects.     

Amino acid uptake by isolated renal brush border membrane vesicles in various buffers

The uptake of amino acids by isolated rat renal brush border membrane vesicles in a modified Krebs-Ringer bicarbonate buffer and a phosphate buffer was compared to the uptake in the standard membrane vesicle buffer, Tris-Hepes-mannitol. The uptake in the modified Krebs-Ringer bicarbonate buffer was similar to that in the Tris-Hepes-mannitol buffer. Removal of the ionic constituents other than NaCl and NaHCO3 in the modified Krebs-Ringer bicarbonate buffer (KCl, CaCl2, KH2PO4 and MgSO4) did not affect the amino acid uptake by the isolated membrane vesicles. The timed uptake of proline under sodium gradient conditions in a phosphate buffer had a markedly dampened overshoot. Kinetic analysis of the initial rate of proline uptake in a phosphate buffer compared to a Tris-Herpes-mannitol buffer showed two entry systems for proline in each buffer with similar Km values, but the maximal rate of transport (V) for each system in the phosphate buffer was much lower than that in the Tris-Hepes-mannitol buffer. From these data, phosphate buffer does not appear to be a suitable medium for the study of amino acid uptake by isolated brush border membrane vesicles.     

A target for cholesterol absorption inhibitors in the enterocyte brush border membrane

Uptake of cholesterol by the intestinal absorptive epithelium can be selectively blocked by specific small molecules, like the sterol glycoside, L-166,143. Furthermore, (3)H-labeled L-166,143 administered orally to hamsters binds specifically to the intestinal mucosa, suggesting the existence of a cholesterol transporter. Using autoradiography, the binding site of (3)H-L-166,143 in the hamster small intestine was localized to the very apical aspect of the absorptive epithelial cells. Label was competed by non-radioactive L-166,143 and two structurally distinct cholesterol absorption inhibitors, suggesting a common site of action for these compounds. L-166,143 blocked uptake of (3)H-cholesterol into enterocytes in vivo, as demonstrated by autoradiography, suggesting that it inhibits a very early step of cholesterol absorption, incorporation into the brush border membrane. This conclusion was confirmed by studies in which intestinal brush borders were isolated from hamsters dosed with (3)H-cholesterol in the presence or absence of L-166,143. Uptake of (3)H-cholesterol into the membranes was substantially inhibited by the compound. In contrast, an inhibitor of acyl CoA:cholesterol acyltransferase, did not affect uptake of (3)H-cholesterol into the brush border membranes. These results strongly support the existence of a specific transporter that facilitates the movement of cholesterol from bile acid micelles into the brush border membranes of enterocytes.     

On the preparation and some properties of closed membrane vesicles from hog duodenal and jejunal brush border

Closed and nearly spherical vesicles were obtained from both hog duodenum and jejunum after mucosa homogenization in the absence of EDTA and a series of fractional centrifugations. The vesicles were found to contain large amounts of two of the characteristic enzyme markers of the brush border membrane (aminopeptidase and alkaline phosphatase). They were seen by electron microscopy on thin sections or after negative staining to be composed of an apparently intact, 90–100 Å-thick membrane overlaid by the fuzzy coat and to be partly filled by a fibrous material tentatively identified with the cross-filaments of the microvilli. This filling was not removed by 5 mM EDTA or/and 1 M Tris unless the structure of the vesicles was largely destroyed. Very few empty vesicles were obtained at the end of these treatments.The vesicles from hog duodenum and jejunum were observed to contain nearly 2 molecules of cholesterol for 1 molecule of phospolipids. Specific differences were noted between both types of vesicles at the level of their sugar composition and associated enzyme activities. For instance, the jejunal vesicles contained no sialic acid and no enterokinase. They contain, respectively, 2 and 4 times as much alkaline phosphatase and aminopeptidase as duodenal vesicles.     

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.     

Proteomic analysis of the enterocyte brush border

The brush border domain at the apex of intestinal epithelial cells is the primary site of nutrient absorption in the intestinal tract and the primary surface of interaction with microbes that reside in the lumen. Because the brush border is positioned at such a critical physiological interface, we set out to create a comprehensive list of the proteins that reside in this domain using shotgun mass spectrometry. The resulting proteome contains 646 proteins with diverse functions. In addition to the expected collection of nutrient processing and transport components, we also identified molecules expected to function in the regulation of actin dynamics, membrane bending, and extracellular adhesion. These results provide a foundation for future studies aimed at defining the molecular mechanisms underpinning brush border assembly and function.     

Transport ofl-cysteine by rat renal brush border membrane vesicles

Brush border membranes were isolated from rat renal cortex by a divalent cation precipitation method. L-35S-cysteine uptake into the vesicles was measured by a rapid filtration method. Only minimal binding of the amino acid to the vesicles was observed. Sodium stimulates L-cysteine uptake specifically. Anion replacement experiments, experiments in the presence of potassium/valinomycin-induced diffusion potential as well as experiments with a potential-sensitive fluorescent dye document an electrogenic sodium-dependent uptake mechanism for L-cysteine. Tracer replacement experiments as well as the fluorescence experiments indicate a preferential transport of L-cysteine. Transport of L-cysteine is inhibited by L-alanine and L-phenylalanine but not by L-glutamic acid and the L-basic amino acids. Initial, linear influx kinetics provide evidence for the existence of two transport sites. The results suggest (a) sodium-dependent mechanism(s) for L-cysteine shared by other neutral amino acids.     

Multiple transport pathways for neutral amino acids in rabbit jejunal brush border vesicles

Amino acids enter rabbit jejunal brush border membrane vesicles via three major transport systems: (1) simple passive diffusion; (2) Na-independent carriers; and (3) Na-dependent carriers. The passive permeability sequence of amino acids is very similar to that observed in other studies involving natural and artificial membranes. Based on uptake kinetics and cross-inhibition profiles, at least two Na-independent and three Na-dependent carrier-mediated pathways exist. One Na-independent pathway, similar to the classical L system, favors neutral amino acids, while the other pathway favors dibasic amino acids such as lysine. One Na-dependent pathway primarily serves neutral L-amino acids including 2-amino-2-norbornanecarboxylic acid hemihydrate (BCH), but not beta-alanine or alpha-methylaminoisobutyric acid (MeAIB). Another Na-dependent route favors phenylalanine and methionine, while the third pathway is selective for imino acids and MeAIB. Li is unable to substitute for Na in these systems. Cross-inhibition profiles indicated that none of the Na-dependent systems conform to classical A or ACS paradigms. Other notable features of jejunal brush border vesicles include (1) no beta-alanine carrier, and (2) no major proline/glycine interactions.