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

Abstract

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 (IP(20), 10(-7) M) or an analog of cAMP (DB-cAMP, 3 x 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 Ca(2+)/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.

Thromboxane A2 Receptor‐Mediated Cell Proliferation, Survival and Gene Expression in Oligodendrocytes

“…Inhibitor concentrations are in agreement with the concentrations previously used by other investigators to specifically inhibit these kinases in cell culture systems (Khorchid et al 1999;Gao et al 2001;Abad et al 2002;Salvi et al 2002). After the treatments, the cells were rinsed with ice-cold PBS and lysed in buffer (20 mM Tris, pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM glycerolphosphate, 1 mM Na 3 VO 4 , 1lg/mL leupeptin and 1 mM PMSF) to extract total cellular protein.…”

“…Signaling pathway of TP-mediated ERK1/2 activation in OPCs and OLGs Specific kinase inhibitors have been used to study the involvement of different kinases in the signaling mechanisms of various cells (Khorchid et al 1999;Gao et al 2001;Abad et al 2002;Salvi et al 2002). To investigate the signaling pathway of TP-mediated ERK1/2 activation in primary OPCs and OLGs, experiments were carried out in the presence of these specific kinase inhibitors.…”

Effect of lipopolysaccharide on D-fructose transport across rabbit jejunum

“…Previous studies in our laboratory have shown that adding LPS to intestinal tissue inhibits Na + -dependent L-leucine intestinal absorption and this effect is related to calcium and involves protein kinase C and calmodulin protein [15,17].…”

Inhibitory effect of TNF‐α on the intestinal absorption of galactose

“…Among the toxins produced by these agents, LPS stimulates the synthesis of inflammatory mediators such as cytokines [Damas et al, 1997]. In previous studies, we reported that LPS inhibits the intestinal transport of L-leucine and D-fructose both when the toxin is directly added to the intestinal tissue [Abad et al, 2001b[Abad et al, , 2002aGarcía-Herrera et al, 2003] or i.v. administered [Abad et al, 2001a].…”

“…Although little is known about the intestinal absorption of nutrients during sepsis, in previous in vivo and in vitro studies, we reported that TNF-a and LPS are able to inhibit the intestinal absorption of L-leucine and D-fructose across the jejunum in rabbits [Abad et al, 2001a[Abad et al, ,b, 2002aGarcía-Herrera et al, 2004].…”