Jejunal Brush-border Vesicles Research Articles

Reduced Folate Carrier: Tissue Distribution and Effects of Chronic Ethanol Intake in the Micropig

Folate deficiency is common in alcoholic patients, in part due to abnormal transport across membranes relevant to folate homeostasis. The reduced folate carrier (RFC) transports monoglutamyl folates across tissue membranes and could be affected by chronic exposure to ethanol. The micropig model is suitable to study the effect of alcoholism on RFC and folate transport across membranes. The membrane transport of [3H]-folic acid was measured by a vacuum filtration method in jejunal brush border (JBB), liver plasma membrane (LPM), and kidney brush border (KBB) membranes vesicles from micropigs fed control or 40% ethanol diets for 12 months. RFC transcripts were analyzed by reverse transcription polymerase chain reaction in jejunal mucosa, liver, and kidney from the same animals. When we compared results from three relevant membranes in control animals, the transport of [3H]-folic acid was highest in LPM, 3-fold lower in KBB (p < 0.001), and 6-fold lower in JBB (p < 0.001). The concentration of RFC transcripts per total RNA was greatest in liver, followed by kidney and jejunum. The transport of [3H]-folic acid by JBB vesicles from chronic ethanol-fed animals exhibited 2-fold lower Km and Vmax (p < 0.05), whereas there was no ethanol effect on the Vmax of [3H]-folic acid transport by LPM or KBB. RFC transcript levels were 10-fold lower in jejunal mucosa from ethanol-fed animals than in control-fed animals (p < 0.005). Although our findings demonstrate different RFC transcript amounts and transport efficiencies among tissues, the present studies suggest that chronic ethanol exposure decreases the intestinal absorption of folic acid by altering the expression of RFC and consequently its transport kinetics in JBB. These findings provide a mechanism for the clinical finding of reduced folic acid absorption in chronic alcoholics.

Distinction between chloride-dependent transport systems for taurine and beta-alanine in rabbit ileum.

This study describes the influx of taurine and beta-alanine across the brush-border membrane of rabbit distal ileum. The kinetics of JmcTau [concentration at which half-maximal activation occurs (K1/2) = 41 microM and Jmax = 24 nmol.cm-2.h-1] are consistent with the kinetics of taurine uptake by jejunal brush-border vesicles. The taurine carrier differs from the beta-alanine carrier by being insensitive to leucine inhibition and by the jejunoileal variation of influx along the small intestine. The K1/2 for sodium and chloride activation of the beta-alanine carrier (48 and 8 mM, respectively) differ markedly from the values reported for the taurine carrier. In addition, taurine is not transported by the beta-alanine carrier. Thus the study demonstrates that the taurine and beta-alanine carriers are separate entities, and it adds to the imino and the taurine carriers the beta-alanine carrier as a third chloride-dependent intestinal transporter of amino acids.

The rabbit jejunal ‘imino carrier’ and the ileal ‘imino acid carrier’ describe the same epithelial function

For rabbit jejunal brush border vesicles an ‘imino carrier’ has been defined as a sodium-depedent, alanine-resistant transporter of cyclic imino acids, while for intact rabbit jejunal and ileal epithelia an ‘imino acid carrier’ has been defined as a sodium-dependent transporter of both aliphatic and cyclic imino acids. This study on intact rabbit intestine examines whether these two terms describe the same epithelial function. The K 1 2 Pro and the K i Pro against J mc MeAIB are identical and so are K 1 2 MeAIB and K i MeAIB against J mc Pro. Likewise, K i Leu is tha same against the transport of both proline and MeAIB. Is is, therefore, concluded that the terms ‘imino carrier’ and ‘imino acid carrier’ describe the same epithelial function: A sodium-dependent, relatively high afinity, saturable transporter of both aliphatic and cyclic imino acids. Estimates of the apparent affinity and inhibitory constants for MeAIB, proline and leucine confirm that the jejuno-ileal variation of amino acid transport along the rabbit small intestine is a variation of maximal transport capacity.

Improved stability of rabbit and rat intestinal brush border membrane vesicles using phospholipase inhibitors

The initial rates of Na +-dependent d-aspartate and d-glucose uptakes were shown to decline from the time of resuspension of brush border membrane vesicles isolated from rabbit and rat jejunum by standard divalent cation precipitation procedures. The former were however more stable than the latter and followed quite closely the decrease in the intravesicular volume, thus suggeting that the loss of transport activity may involve both nonspecific opening of the vesicles and either direct or indirect specific inactivation of the transporters. Uptake rates for both substrates did tend to stabilize at 6–24 h from resuspension, however this final ‘next day’ uptake activity was too low to be of practical use in kinetic studies. Freeezing aliquots of rabbit jejunal vesicles in liquid N 2 until the time of assay resulted in complete stabilization of d-glucose uptake. A modified homogenate buffer designed to inhibit a broad spectrum of phospholipase activities resulted in a partial stabilization of glucose transport by rabbit jejunal vesicles with, on average, an over 6-fold enrichment in the ‘next day’ stable specific activity of uptake as compared to unfrozen vesicles. The modified homogenate buffer also improved the stability and the ‘next day’ specific activities of d-glucose uptake in rat jejunal brush border vesicles and d-aspartic acid uptake in rabbit jejunal vesicles. It also completely stabilized the intravesicular volume in the latter preparation. An evaluation of the kinetic parameters of Na +-dependent d-glucose transport in rabbit vesicles prepared from either the standard homogenate media and frozen in liquid N 2 or the modified media and allowed to stabilizeovernight, revealed a single transport system with a K m of 0.31–0.32 mM as the best model to fit the data. As such the modifications to the homogenate media do not appear to effect the functional properties of d-glucose transport in the membrane. While being less efficient in stabilizing the vesicles than the rapid freezing protocol, it is shown that the modified homogenate should however be preferred when dealing with slowly permeant ions like choline since it provides in this case the only alternative to reliable measurement of uptake rates across a stable and equilibrated vesicle preparation.

Effects of chloride conductance inhibitors on fluid secretion into ligated ileal and jejunal loops in pigs

SUMMARY Compounds that prevent chloride transport in membrane vesicles have been tested for in vivo activity against the effects of intestinal secretory agents. Chloride channel blockers including diphenylamine-2-carboxylate, 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonate, 5-nitro-2-(2-phenylethylamino)benzoic acid, and α-phenylcinnamic acid were tested for effects on jejunal or ileal secretion in weanling pigs. Secretion was studied in ligated intestinal loops in a control state, during exposure to secretory concentrations of theophylline, and after prior treatment with cholera toxin. Increases in net fluid flux induced by either theophylline or cholera toxin were not prevented by adding chloride channel blockers into the intestinal lumen. Channel blocker concentrations that reduced chloride transport by &gt; 50% in pig jejunal brush border vesicles did not cause significant changes in unidirectional blood to lumen chloride flux measured in situ. Several routes of administration of the specific chloride channel blocker α-phenylcinnamate failed to reduce fluid secretion induced by theophylline. Chloride channel blocker effectiveness appears to be significantly different between in vitro and in vivo experimental models. In contrast to the chloride channel blockers, loperamide significantly reduced net fluid and chloride flux in ileal loops secreting fluid in response to theophylline. Antagonism of the production or actions of second messenger by loperamide was more effective than the chloride channel blockers in reducing conductive chloride transport associated with intestinal secretion.

Lysine and methionine transport by bovine jejunal and ileal brush border membrane vesicles.

Lysine (LYS) and methionine (MET) transport were studied using brush border membrane vesicles from bovine jejunal and ileal tissues. Total transport of LYS and MET was divided into mediated and diffusion components. Mediated uptake was further divided into sodium (Na)-dependent and Na-independent systems. Total LYS and MET uptake by ileal brush border (BB) vesicles tended to be higher than that by jejunal BB vesicles at all concentrations evaluated but differences were significant (P less than .05) at 2.5 and 7.5 mM for LYS and 5, 12.5 and 15 mM for MET. The greater capacity of ileal BB vesicles appeared to be due to the Na-dependent component of LYS uptake and the diffusion component of MET uptake. Transporters had less affinity but higher capacity than for LYS transport in both ileal and jejunal tissue. Methionine transport was greater (P less than .05) than LYS transport in both ileal and jejunal BB vesicles when the initial amino acid concentration was 7.5 mM. But when the initial amino acid concentration was 1.25 mM, MET uptake was greater (P less than .13) than LYS uptake in jejunal, but not in ileal, BB vesicles. The relative contribution of mediated and diffusion uptake systems to total MET and LYS uptake was found to be dependent on substrate concentration. Both intestinal site and substrate concentration influenced the contributions of Na-dependent, Na-independent and diffusion systems to total methionine and lysine uptake.

Diabetes mellitus and the sodium electrochemical gradient across the brush border membrane of rat intestinal enterocytes.

The effects of chronic diabetes mellitus of 4-5 weeks duration on the potential difference across the brush border membrane of rat small intestine (Vm) and on Na+-dependent uptake of D-glucose by jejunal brush border vesicles have been studied. Diabetes increased Vm in the jejunum from a mean value of -47.2 mV in control tissue to -57.4 mV in diabetic tissue (P less than 0.001) but was without effect on ileal Vm. Measurements of Vm during ion-substitution experiments revealed that the conductance of Na+ of the jejunal brush border was reduced by diabetes, whilst K+ and Cl- permeabilities were unaltered. Uptake studies using brush border vesicles and an Na+-electrochemical gradient showed that diabetes caused a 56% increase in the initial rate of uptake of D-glucose but was without effect on the peak to equilibrium ratio. Taken together, data from these two studies suggest that the lower Na+ permeability of the brush border in diabetes enhances the electrical and chemical driving force for active Na+-dependent uptake of glucose by reducing glucose-independent movement of Na+ across this membrane. Finally, the possible humoral factors involved in this response to diabetes are discussed.

Activation of chloride conductance in pig jejunal brush border vesicles.

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, AsO4(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.

Calcium transport affinity, ion competition and cholera toxin effects on cytosolic Ca concentration.

The physiological relevance of an apparent ionophore activity of cholera toxin towards Ca2+ has been examined in several different systems designed to measure affinity, specificity, rates of ion transfer, and effects on intracellular ion concentrations. Half-maximal transfer rates across porcine jejunal brush-border vesicles were obtained at a concentration of 0.20 microM Ca2+. When examined in the presence of competing ions the transfer process was blocked by very low concentrations of La3+ or Cd2+, Sr2+, Ba2+ and Mg2+ were relatively inefficient competitors for Ca2+ transport mediated by cholera toxin. The relative affinities observed would be compatible with a selectivity for Ca2+ transfer at physiological ion concentrations, as well as an inhibition of this ionophore activity by recognized antagonists of cholera toxin such as lanthanum ions. Entry rates of Ca2+ into brush-border vesicles exposed to cholera toxin were large enough to accelerate the collapse of a Ca2+ gradient generated by endogenous Ca, Mg-ATPase activity. The treatment of isolated jejunal enterocytes with cholera toxin caused a significant elevation in cytosolic Ca2+ concentrations as measured by Quin-2 fluorescence. This effect was specifically prevented by prior exposure of the cholera toxin to excess ganglioside GM1. We conclude that cholera toxin has many of the properties required for promoting transmembranes Ca2+ movement in membrane vesicles and appears to be an effective Ca2+ ionophore in isolated mammalian cells.

Critical role of ring structure in the differential uptake of cholesterol and plant sterols by membrane preparations in vitro.

To determine the role of the ring structure in the differential absorption of sterols, we have used rat jejunal brush border vesicles and erythrocytes to examine the uptake of cholesterol, campesterol, and sitosterol following successive chemical degradations of rings A and B. The cell and membrane preparations were incubated with the sterols and sterol analogues (about 30 micromolar each) dissolved in 7 mM sodium taurocholate and 0.6 mM egg phospholipid. The uptake of the analogues was analyzed by high performance liquid chromatography and capillary gas--liquid chromatography. In both membrane preparations, the uptake of the 7-dehydroanalogues of cholesterol, campesterol, and sitosterol was linear with time. 7-Dehydrocholesterol was absorbed 4-5 times faster than 7-dehydrositosterol by both preparations. The uptake of the campesterol analogue was intermediate between that of the analogues of cholesterol and sitosterol at all time points. Following conversion of the 7-dehydrosterols to their calciferol derivatives, the 27-carbon sterols were absorbed only 1.9 and 1.4 times faster than those of the 29-carbon sterols by the erythrocyte and brush border membranes, respectively. A similar degree of selectivity was expressed in the erythrocytes during the uptake of a steroid series possessing keto-4-ene ring system. Complete oxidation of the calciferol derivatives to the des-AB-8-ones resulted in a total loss of discrimination among the various side-chain homologues during absorption from micellar solutions. It is concluded that the selective absorption of animal and plant sterols depends upon the presence of a ring system having the bulk of the cholestane nucleus, although not necessarily a rigid or planar one containing a hydroxyl group.

Kinetics of sodium-dependent solute transport by rabbit renal and jejunal brush-border vesicles using a fluorescent dye.

The kinetics of Na-coupled solute transport by renal and jejunal brush-border vesicles in the rabbit were examined using the potential-sensitive fluorescent dye diS-C3-(5). All organic solutes known to be transported across these membranes by Na-coupled mechanisms increase the fluorescence of the dye in the presence of Na, but not K. An increase in fluorescence (delta F) corresponds to a depolarization of the electrical potential difference (5-60 mV) across the brush-border membrane in the intact cell. delta F was independent of the valency of the transported solute. The fluorescence response was saturable, and for twelve solutes the Kf, i.e. the concentration of the substrate generating 50% of the maximal response, agreed quite closely with the Kt values reported from tracer studies. For six solutes increasing the Na concentration decreased Kf, and this agrees with the effect of Na on the kinetics of succinate transport in renal vesicles. We conclude that D-glucose, neutral amino acids and imino acids are co-transported with Na across both renal and jejunal brush-border membranes, and that carboxylic acids, beta-amino acids, and dibasic amino acids are co-transported with Na across the renal, but not jejunal, membranes.

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.