Rabbit Jejunal Brush-border Membrane Vesicles Research Articles

Effect of ischemia on intestinal permeability of lipopolysaccharides.

The enteral absorption of endotoxin (lipopolysaccharide, LPS) was studied in vitro and in vivo. The absorption of fluorescence (FITC) labelled LPS was investigated by uptake studies with rabbit jejunal brush-border membrane vesicles (BBMV), the human intestinal cell line Caco-2 and in rats. FITC-LPS from Salmonella typhimurium was taken up into BBMV in a dose-dependent manner. Uptake was neither pH- nor Na+-dependent, nor could it be inhibited by unlabelled LPS. 74.5% of vesicle-associated fluorescence was due to adhesion to the vesicular membranes, 25.5% was taken up into an osmotically-sensitive space. Transport of LPS across Caco-2 cell monolayers was dose-dependent. The permeation rate increased significantly under ischemic conditions, i.e. when the cells were incubated under oxygen-depleted conditions. However, no significant differences in transepithelial electrical resistances were observed between oxygen depleted and control cells. The release of lactate dehydrogenase was only marginally different from control cells, indicating cell integrity. In situ, after gut ischemia, a significantly increased uptake of fluorescent LPS was observed by fluorescence laser scanning microscopy. Conclusion LPS is taken up by the intestinal mucosa, predominantly by passive transcellular diffusion. Under ischemic conditions, the permeability of LPS is increased mainly by an enhanced paracellular permeability and epithelial destruction. The findings partly explain the clinically observed development of multiorgan system failure after temporary malperfusion of the gut during major vascular surgery or thromboembolism.

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

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

Nicotinic acid transport mediated by pH-dependent anion antiporter and proton cotransporter in rabbit intestinal brush-border membrane.

In order to determine whether the vitamin nicotinic acid is absorbed via an anion antiporter, intestinal epithelial cell membrane transport mechanisms for nicotinic acid were characterized using isolated rabbit jejunal brush-border membrane vesicles. The uptake of nicotinic acid by the membrane vesicles showed an overshoot phenomenon in the presence of an outwardly directed bicarbonate gradient or an inwardly directed proton gradient and the uptakes were two times and six times greater, respectively, than that in the absence of any ion gradient. The bicarbonate-dependent initial uptake of nicotinic acid was increased at acidic pH, showing pH-dependent transport activity. An inhibitor of anion transport, 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid, specifically reduced bicarbonate-dependent transport of nicotinic acid. The initial uptakes of nicotinic acid via the anion antiporter and the proton cotransporter were specifically inhibited by monocarboxylic acids such as acetic acid, benzoic acid, D- and L-lactic acid, pravastatin and valproic acid, but not by di- or tricarboxylic acids, bile acids or amino acids. Nicotinic acid uptake activity was, furthermore, expressed in a Xenopus laevis oocyte system after injection of messenger RNA (mRNA) derived from rabbit intestinal epithelial cells. These observations demonstrate that nicotinic acid is absorbed by two independent active transport mechanisms from small intestine, i.e. a proton cotransporter and an anion antiporter. The pH-dependence observed in the intestinal absorption of nicotinic acid might, therefore, be ascribed partly to pH-sensitive and partly to carrier-mediated transport mechanisms in the brush-border membrane.

Heterotropic effects of dipolar amino acids on the activity of the anionic amino acid transport system X-AG in rabbit jejunal brush-border membrane vesicles

D-Aspartic acid was used as a specific substrate to evaluate the effects of dipolar amino acids on the high affinity anionic amino acid transport system X-AG in rabbit jejunal brush-border membrane vesicles. At pH 6, increasing L-phenylalanine concentrations caused a saturable activation of 0.05 mM D-aspartic acid uptake (Ka = 2.4 mM), and a saturating concentration of effector increased the Vmax of transport (2.6-fold) without any significant effect on the Km. At pH 8, however, a complex activation/inhibition curve was obtained with increasing L-phenylalanine concentrations, and a saturating concentration of effector increased both the Vmax (1.5-fold) and Km (2.1-fold) for transport. Increasing concentrations of L-valine, L-isoleucine, L-methionine, and L-threonine also showed complex activation/inhibition curves of D-aspartic acid uptake at both pH 6 and 8. The maximum level of activation, the plateau reached at saturating concentrations, and the concentration of effector producing either maximum activation or inhibition were, however, different at these two pH values. By using an optimum concentration of 10 mM L-valine at pH 6, the absence of trans-activation and of further activation by a cis-gradient of effector could be demonstrated. These results suggest that two allosteric sites directly accessible from the external medium are responsible for the heterotropic activation of intestinal system X-AG by dipolar amino acids and that, under physiological conditions, such effects might compensate for the lack of specificity of the neutral brush-border system.

Transport des Hydroxyanalogons von Leucin in Bürstensaum-Membranvesikel des Kaninchendünndarms

Hydroxy analogues of essential amino acids can be used in clinical nutrition to minimize nitrogen intake. In this study intestinal uptake of L-leucine hydroxy analogue into rabbit jejunal brush-border membrane vesicles was investigated. An inward directed H(+)-gradient was a driving force of uptake (pHoutside = 6.0; pHinside = 7.5) and led to a transient accumulation. The saturable system has a apparent transport constant Kt = 15.4 mM. By trans stimulation experiments it could be shown that both D- and L-stereoisomers of hydroxy analogues of branched chain amino acids as well as L-lactate share with the same H(+)-driven uptake system.

Transport of l-leucine hydroxy analogue and l-lactate in rabbit small-intestinal brush-border membrane vesicles

Substitution of the alpha-amino group of amino acids by hydroxyl groups yields hydroxy analogues (HA), which have been ascribed beneficial effects in nitrogen-sparing diets for uremic patients. In this study, intestinal uptake of L-leucine HA (L-LeuHA) and L-lactate into rabbit jejunal brush-border membrane vesicles was investigated. An inward-directed H+ or Na+ gradient stimulated uptake of both labelled substrates in a voltage-clamped assay. The H+ gradient was the major driving force of uptake as compared with the Na+ gradient, and it led to a transient accumulation of both L-LeuHA and L-lactate. The proton ionophore carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) reduced the initial H(+)-gradient-driven uptake rates of both substrates, but was without effect on Na(+)-gradient-driven uptakes. The H(+)-gradient-driven L-LeuHA uptake was saturable (apparent Kt = 15.4 mM). Alpha-HA of L-leucine, L-isoleucine, L-valine, D-leucine, D-valine or L-lactate inhibited the H(+)-gradient-driven L-LeuHA or L-lactate uptakes whereas free branched-chain amino acids had no effect. Preloading the vesicles with one of the L- or D-HA of branched-chain amino acids or with L-lactate stimulated tracer L-LeuHA and also tracer L-lactate uptakes in the presence of a H+ gradient. It is concluded that H(+)-gradient-driven transport of L- and D-stereoisomeric HA of branched-chain amino acids as well as of L-lactate across rabbit intestinal brush-border membranes is mediated by the same carrier. Furthermore, there exists a Na+ gradient-driven L-lactate transport system in the rabbit intestinal brush-border membrane.

Highly permeant anions and glucose uptake as an alternative for quantitative generation and estimation of membrane potential differences in brush-border membrane vesicles

We have analyzed the combined utilization of highly permeant anions to induce membrane diffusion potentials and glucose uptake to probe the created potentials as a new approach to quantitative generation and estimation of membrane potential differences in vesicle studies. Rabbit jejunal brush-border membrane vesicles were used in our experiments so that membrane potential differences can be calculated from the Goldman-Hodgkin-Katz equation with the relative ion permeabilities recently reported for this preparation (Gunther, R.D., Schell, R.E. and Wright, E.M. (1984) J. Membrane Biol. 78, 119–127) or approximated by the Nernst potential for the anion. Iodide was selected as the highly permeant anion after showing its absence of effect on glucose uptake with equal concentrations of Na + inside and outside the vesicles and the membrane potential clamped to zero with gramicidin D. Membrane potential was varied by altering the intra- and extravesicular iodide concentrations while keeping isosmolarity and isotonicity constant by chloride replacement. In these conditions, glucose uptake was sensitive and correlated to the expected membrane potentials. Moreover, a linear relationship between the log initial rate of glucose transport and membrane potential differences could be established. This linear relationship was quite insensitive to inside replacement of choline by potassium and to pH variations in the incubation medium, thus showing the reproducibility and the versatility of the method and the adequacy of glucose uptake as a probe for membrane potentials. However, no information can be gained on the stoichiometry of the Na +-glucose transporter as the slope of the straight line depends on both the charge carried by the fully loaded carrier and the point in the electric field at which the transition state of the carrier from cis to trans occurs. This new approach was compared with the more conventional one using valinomycin-induced K +-diffusion potentials and the Nernst potential for potassium as means for creating and estimating membrane potential differences. Both techniques were not equivalent, as linear relationships showing smaller slopes and sensitivity to pH were recorded with the latter. These differences are compatible with a potassium permeability in the presence of valinomycin that is lower than generally assumed, at least when compared to the permeability of the other ions present in the incubation medium, it is concluded that our new procedure offers significant advantages over more classical approaches, particularly to test for membrane potential dependency of sodium cotransport systems in which cations have been implicated in the transport mechanism. This method can also be extended to other vesicle preparations by properly selecting an highly permeant anion. In the absence of knowledge upon the (relative) ion permeabilities of the membrane, an estimation of membrane potential differences by the Nernst potential for this anion should prove as good an approximation as the Nernst potential for potassium in the presence of valinomycin.

Membrane potential dependency of glutamic acid transport in rabbit jejunal brush-border membrane vesicles: K + and H + effects

We have applied our recently developed approach for quantitative generation and estimation of membrane potential differences (Berteloot, A. (1986) Biochim. Biophys. Acta 857, 180–188) to the reevaluation of glutamic acid transport rheogenicity in rabbit jejunal brush-border membrane vesicles. Membrane diffusion-potentials were created by altering iodide concentrations in the intra- and extravesicular compartments while keeping isosmolarity, isotonicity and ionic strength constant by chloride replacement. The known value of ion permeabilities relative to sodium in this preparation also allows calculation of membrane potential differences using the Goldman-Hodgkin-Katz equation. This strategy appears superior to more classical methods involving ionophore-induced membrane diffusion-potentials of protons or potassium as both cations have been shown to participate in the transport mechanism. In this paper, we demonstrate that this approach is perfectly suitable for the investigation of membrane potential dependency of glutamic acid transport as our results showed that chloride replacement by iodide did not affect uptake in vesicles with membrane potential clamped to zero by gramicidin D (sodium conditions) or by gramicidin D plus valimonycin (sodium + potassium conditions). The method thus allows to dissociate membrane potential effects from possible effects that might be introduced by altering the anion species. In these conditions, our studies clearly demonstrate that glutamic acid uptake, whether analyzed over a 1 min time scale or under initial rate conditions, was sensitive to membrane potential differences. However, our results also show that the electrogenicity of the transport system varied depending upon the intravesicular presence or absence of potassium, its presence stimulating the membrane potential dependency of uptake. This effect is modulated by the internal pH and it is concluded that inside H + and K + are not equivalent as countertransported cations. The external pH also seems to modulate the response to potential by acting on the fully loaded form(s) of the transporter. The possibility that outside H + competes for (an) external Na + binding site(s) and/or precludes the attachment of (an) extra sodium ion(s) should be considered.