M.J. Rodriguez-Yoldi

Effect of lipopolysaccharide on D-fructose transport across rabbit jejunum

Abstract:Objective: To investigate alterations in the transport of D-fructose across the rabbit jejunum when the gut is exposed in vitro to lipopolysaccharide (LPS), an endotoxin causative agent of sepsis. Materials and methods: D-fructose intestinal transport was assesed employing three techniques: sugar uptake measurements in rings of everted jejunum (μmol/D-fructose/ml cell water), transepithelial flux measurements in Ussing-type chambers (μmol D-fructose/cm2/h) and transport assays in preparation of brush border membrane vesicles (pmoles D-fructose/mg protein). Samples were taken from the bathing solution and from the extracts of the tissue for radioactivity counting. Results: Adding LPS (3 μg/ml) to tissue decreased the uptake and mucosal to serosal flux of 5 mM D-fructose across the enterocyte. LPS did not modify sugar uptake across brush border membrane vesicles. The inhibitory effect of LPS was suppressed by W-13 (5 × 10–6 M), a Ca-calmodulin antagonist, and staurosporine (10–7 and 10–6 M) and GF-109203X (10–6 M) a nonselective and selective protein kinase C (PKC) inhibitor respectively. Tumor Necrosis Factor (TNF-α), an immunoregulatory cytokine involved in septic responses occurring during bacterial infection at concentrations 3 × 10–4 to 3 μg/ml, did not affect the sugar transport. Conclusions: LPS can inhibit the intestinal uptake of D-fructose across the rabbit jejunum in vitro by intracellular processes related to PKC and calmodulin protein.

Effect of lipopolysaccharide on small intestinal L-leucine transport in rabbit

In the present study, we have investigated whether the lipopolysaccharide (LPS) endotoxin from Escherichia coli is able to alter the jejunal transport of l-leucine when the tissue is exposed to endotoxin. The results have shown that the LPS at 3 × 10-5 μg/ml decreases the uptake of l-leucine into the enterocyte, as well as the mucosal to serosal flux of l-leucine. The secretagogue effect of LPS on the gut did not affect the inhibitory effect of LPS on the intestinal absorption of the amino acid. The endotoxin did not modify amino acid diffusion across the intestinal epithelium. However, from the mediated transport, only the Na+-dependent transport system was affected by LPS with a diminution of the transporter affinity (the apparent Km was increased). In addition, we found a reduction of the Na+, K+-ATPase activity, which could explain the l-leucine Na+-dependent transport inhibition.

5-HT RECEPTOR SUBTYPES INVOLVED IN THE SEROTONIN-INDUCED INHIBITION OF L-LEUCINE ABSORPTION IN RABBIT JEJUNUM

The aim of the present study was to determine the 5-HT receptor subtypes involved in the serotonin-induced inhibition of L-leucine absorption across rabbit jejunum in vitro. A number of agonists and antagonists were used to characterize the receptors through which serotonin inhibits this absorption. The results show that 2.5x10(-6) M 5-HT inhibits the amino acid absorption by about 20%. The 5-HT receptor agonists, alpha-methyl-5-HT (5-HT2), 2-methyl-5-HT (5-HT3) and zacopride (5-HT4) at concentrations 2.5x10(-6) and 2.5x10(-5) M produced 10-30% inhibition on L-leucine intestinal absorption. 5-carboxyamidotryptamine (5-HT1) did not produce any inhibition. The 5-HT antagonists, GR 113808A (5-HT4) at 2.5x10(-6) M and ritanserin (5-HT2) and ondansetron (5-HT3) at 2.5x10(-5) M completely blocked the effect of 5-HT. However, methiothepin (5-HT1) did not produce any effect on serotonin action in the intestinal absorption of amino acid. It can be concluded that 5-HT2, 5-HT3 and 5-HT4 receptors could mediate inhibition of L-leucine absorption across rabbit jejunum.

Cellular mechanism underlying LPS-induced inhibition of in vitro L-leucine transport across rabbit jejunum:

Lipopolysaccharide(LPS) is a known causative agent of sepsis. In previous studies, we have shown that it reduces L-leucine mediated transport across the rabbit jejunum by about 30%. In this study, the mechanism(s) of LPS inhibition on amino acid transport were analysed in detail. LPS did not inhibit L-leucine transport across brush border membrane vesicles, suggesting the need for an intracellular step. The inhibitory effect of LPS was not altered by the addition of protein kinase A (PKA) inhibitor (IP20, 10—7 M) or an analog of cAMP (DB-cAMP, 3 × 10—4 M), indicating that the PKA signal transduction pathway was not involved in the LPS effect. However, the inhibitory effect of LPS was suppressed by trifluoroperazine (10—7 M), a Ca2+/calmodulin inhibitor and staurosporine (10—7 M), an protein kinase C (PKC) inhibitor. Likewise, LPS inhibition disappeared in media without calcium. These results suggest that LPS could inhibit the intestinal uptake of L-leucine across the small intestine in vitro by intracellular processes related to calcium, involving PKC and calmodulin protein.

The administration of lipopolysaccharide, in vivo, induces alteration in L-leucine intestinal absorption.

The objective of the present study was to determine the alterations in L-leucine intestinal uptake by intravenous administration of Lipopolysaccharide (LPS), which is a constituent of gram negative bacterial, causative agent of sepsis. The amino acid absorption in LPS treated rabbits was reduced compared to the control animals. The LPS effect on the amino acid uptake was due to an inhibition of the Na+-dependent system of transport, through both reduction of the apparent capacity transport (Vmax) and diminution of the Na+/K-ATPase activity. The results have also shown that the LPS decreases the mucosal to serosal transepithelial flux and the transport across brush border membrane vesicles of L-leucine. The study of possible intracellular mechanisms implicated in the LPS effect, showed that the second messengers calcium, protein kinase C and c-AMP did not play any role in this effect. However, the absence of ion chloride in the incubation medium removes the LPS inhibition and the intracellular tissue water was affected by the LPS treatment. Therefore, the inhibition in the L-leucine intestinal absorption, by intravenous administration of LPS, could be mainly produced by the secretagogue action of this endotoxin on the gut.

Tumor necrosis factor-α mediates inhibitory effect of lipopolysaccharide on L-leucine intestinal uptake

Tumor necrosis factor-α (TNF-α) has been proposed as an early proximal mediator of many metabolic and physiologic responses during septic shock. We have previously shown that direct addition to tissue (local effect) or intravenous administration (systemic effect) of lipopolysaccharide (LPS) reduces l-leucine absorption across rabbit jejunum. In the present study, we investigated whether the inhibitory effect of LPS on l-leucine intestinal absorption in rabbit is related to TNF-α. The results shown that the addition of TNF-α to tissue does not produce any effect on l-leucine uptake by the enterocyte. When TNF-α was inoculated by intravenous administration, a strong inhibition on the l-leucine uptake (about 40%), mediated by a secretagogue effect on water and Cl-ions was induced. We also found that the LPS intestinal effect induced by intravenous administration, was blocked by a TNF-α antagonist, indicating that TNF-α is a mediator of the LPS systemic effect on l-leucine intestinal uptake inhibition. The study of possible mediators involved in the TNF-α effect showed that nitric oxide and prostaglandins are implicated in the l-leucine intestinal uptake.

Serotonin-induced changes in L-leucine transport across rabbit jejunum

The aim of the present study has been to determine the effect of serotonin (5-HT) on the absorption of L-leucine across the rabbit jejunum. The results show that serotonin significantly diminishes the uptake and steady-state tissue accumulation and the mucosal to serosal flux of L-leucine. This effect does not change with previous intestinal exposure of the mucosa to the 5-HT. Serotonin does not seem to modify amino acid simple diffusion across the intestinal epithelium. The effect on the amino acid uptake is due to an inhibition of the Na(+)-dependent system of transport, mainly through a reduction of the apparent Vmax. Moreover, this hormone, added to the incubation solution, does not affect the L-leucine uptake across brush border membrane vesicles. In presence of trifluoroperazine, TMB-8 and staurosporine, the serotonin effect disappears. These results suggest an effect mediated by intracellular processes related to protein kinase C which inhibit the intestinal absorption of L-leucine.

Action of zinc on enzymatic digestion and intestinal transport of sugar in the rabbit.

The aim of the present work was to study whether zinc chloride added to the drinking water of rabbits affected the intestinal absorption of D-galactose and the activity of sucrase in the jejunum. The results showed that zinc decreased D-galactose absorption in the jejunal tissue. The effect appeared to be due mainly to an action on the active transport of the sugar by the mucosal border of the intestinal epithelium, because the zinc seemed not to affect its diffusion across the intestinal epithelium. Zinc was also shown to inhibit the (Na(+)-Ka+)-ATPase activity of the enterocyte, which might explain the inhibition of the Na(+)-dependent transport of D-galactose. Nevertheless, a possible direct action of the zinc ion on the Na(+)-dependent carrier cannot be discounted. Zinc did not alter the activity of sucrase in the jejunum of the rabbit.

The effect of cytokines on intestinal sugar absorption during sepsis in rabbits.

The endotoxin that triggers an immune response to Gram-negative bacterial infection namely lipopolysaccharide (LPS), is also associated with gastrointestinal abnormalities and induces the release of proinflammatory cytokines such as IL-1 and TNF-α. The main aim of this study was to determine the effect of cytokine release on intestinal D-fructose absorption in LPS-treated rabbits in order to provide information that could be used to understand their septic status. The results obtained, using whole tissue and brush border membrane vesicles from rabbit jejunum, showed that LPS, TNF-α and IL-1β inhibit d-fructose absorption across the jejunum. The effect of LPS is completely reversed by a TNF-α antagonist and partially by a specific IL-1 receptor antagonist (IL-1ra) and disappears completely in the presence of both these cytokine antagonists. Similarly, the effects of TNF-α and LPS were not totally blocked by IL-1ra, whereas the effect of IL-1β disappeared completely in the presence of a TNF antagonist. In summary, these results show that TNF-α and IL-1β could act synergistically on sugar absorption in rabbit with LPS-induced sepsis. In addition, the effects of IL-1β depend on, or are related to TNF-α production since this effect returns to basal (control) levels in the presence of a TNF-α antagonist.

Study of interaction between calcium and zinc on D-galactose intestinal transport.

Zinc is an essential trace element necessary to life. This metal may exert some of its physiological effects by acting directly on cellular membranes, either by altering permeability or by modulating the activity of membrane-bound enzymes. On the other hand, calcium is an essential element in a wide variety of cellular activities. The aim of the present work was to study a possible interaction between zinc and calcium on intestinal transport ofd-galactose in jejunum of rabbit in vitro. In media with Ca2+, when ZnCl2 was present at 0.5 or 1 mM, zinc was found to reduce thed-galactose absorption significantly. In Ca2+-free media, where CaCl2 was omitted and replaced isotonically with choline chloride, the sugar transport was not modified by zinc. Verapamil at 10−6M (blocking mainly Ca2+ transport) did not modify the inhibitory effect of zinc ond-galactose transport. When 10−6M of A 23187 (Ca2+-specific ionophore) was added with/without Ca2+ to the media, ZnCl2 produced no change in sugar transport. These results could suggest a possible interaction of calcium and zinc for the same chemical groups of membrane, which could affect the intestinal absorption of sugars.