Increases in circulating amino acids with in-feed antibiotics correlated with gene expression of intestinal amino acid transporters in piglets.

In-feed antibiotics have been commonly used to promote the growth performance of piglets. The antibiotics can increase protein utilization, but the underlying mechanism is largely unknown. The present study investigated the effects of in-feed antibiotics on intestinal AA transporters and receptors to test the hypothesis that the alteration of circulating AA profiles may be concomitant with the change of intestinal AA transporters and receptors. Sixteen litters of piglets at day 7 started to receive creep feed with (Antibiotic) or without (Control) antibiotic. Piglets were weaned at day 23 after birth, and fed the same diets until day 42. In-feed antibiotics did not affect the BW of 23-day-old (P = 0.248), or 42-day-old piglets (P = 0.089), but increased the weight gain to feed ratio from day 23 to 42 (P = 0.020). At day 42 after birth, antibiotic treatment increased the concentrations of most AAs in serum (P < 0.05), and decreased the concentrations of most AAs in jejunal and ileal digesta. Antibiotics upregulated (P < 0.05) the mRNA expression levels for jejunal AAs transporters (CAT1, EAAC1, ASCT2, y+LAT1), peptide transporters (PepT1), and Na+-K+-ATPase (ATP1A1), and ileal AA transporters (ASCT2, y+LAT1, b0,+AT, and B0AT1), and ATP1A1. The antibiotics also upregulated the mRNA expression of jejunal AAs receptors T1R3 and CaSR, and ileal T1R3. Protein expression levels for jejunal AA transporters (EAAC1, b0,+AT, and ASCT2) and PepT1 were also upregulated. Correlation analysis revealed that the alterations of AA profiles in serum after the in-feed antibiotics were correlated with the upregulations of mRNA expression levels for key AA transporters and receptors in the small intestine. In conclusion, the in-feed antibiotics increased serum level of most AAs and decreased most AAs in the small intestine. These changes correlated with the upregulations of mRNA expression levels for key AA transporters and receptors in the small intestine. The findings provide further insights into the mechanism of in-feed antibiotics, which may provide new framework for designing alternatives to antibiotics in animal feed in the future.

L-Glutamate Enhances Barrier and Antioxidative Functions in Intestinal Porcine Epithelial Cells.

BACKGROUND: L-Glutamate (Glu) is a major amino acid in milk and postweaning diets for mammals (including pigs and human infants). However, effects of Glu on intestinal mucosal barrier and antioxidative functions are unknown. OBJECTIVE: This study tested the hypothesis that Glu may enhance the barrier function of intestinal porcine epithelial cell line 1 (IPEC-1) cells by upregulating the expression of tight junction proteins. METHODS: IPEC-1 cells were cultured with or without Glu in the presence or absence of 1 mmol/L diquat (an oxidant) for indicated time points. Cell numbers, transepithelial electrical resistance (TEER), mRNA, and protein abundance of glutamate transporter, the release of lactate dehydrogenase (LDH), and the abundance of tight junction proteins were determined. RESULTS: Compared with 0 mmol/L Glu, 0.5-, 1-, and 2 mmol/L Glu stimulated (P < 0.05) cell growth by 13-37% at 24 h and 12-34% at 48 h, respectively. In addition, 0.5 mmol/L Glu increased (P < 0.05) TEER (by 58% at 24 h and by 98% at 48 h, respectively). These effects of Glu were associated with increased mRNA abundance of Glu transporter solute carrier family 1 member 1 (SLC1A1) by 30-130% and protein abundance of excitatory amino acid transporter 3 (encoded by SLC1A1) by 19-34%, respectively. In a cell model of oxidative stress induced by 1 mmol/L diquat, 0.5 mmol/L Glu enhanced cell viability, TEER, and membrane integrity (as indicated by the reduced release of LDH) in IPEC-1 cells by increasing the abundance of the tight junction proteins occludin, claudin-3, zonula occludens (ZO)-2, and ZO-3. CONCLUSION: These findings indicate that Glu plays an important role in mucosal barrier function by enhancing cell growth and maintaining membrane integrity in response to oxidative stress.

Chemical activation of a high-affinity glutamate transporter in human erythrocytes and its implications for malaria-parasite-induced glutamate uptake.

Human erythrocytes have a low basal permeability to L-glutamate and are not known to have a functional glutamate transporter. Here, treatment of human erythrocytes with arsenite was shown to induce the uptake of L-glutamate and D-aspartate, but not that of D-glutamate or L-alanine. The majority of the arsenite-induced L-glutamate influx was via a high-affinity, Na(+)-dependent system showing characteristics of members of the "excitatory amino acid transporter" (EAAT) family. Western blots and immunofluorescence assays revealed the presence of a member of this family, EAAT3, on the erythrocyte membrane. Erythrocytes infected with the malaria parasite Plasmodium falciparum take up glutamate from the extracellular environment. Although the majority of uptake is via a low-affinity Na(+)-independent pathway there is, in addition, a high-affinity uptake component, raising the possibility that the parasite activates the host cell glutamate transporter.

Riluzole enhances the activity of glutamate transporters GLAST, GLT1 and EAAC1.

Riluzole exerts a neuroprotective effect through different mechanisms, including action on glutamatergic transmission. We investigated whether this drug affects glutamate transporter-mediated uptake, using clonal cell lines stably expressing the rat glutamate transporters GLAST, GLT1 or EAAC1. We found that riluzole significantly increased glutamate uptake in a dose-dependent manner; kinetic analysis indicated that the apparent affinity of glutamate for the transporters was significantly increased, with similar effects in the three cell lines. This may facilitate the buffering of excessive extracellular glutamate under pathological conditions suggesting that riluzole's neuroprotective action might be partly mediated by its activating effect on glutamate uptake.

Prenatal exposure to the cannabinoid receptor agonist WIN 55,212-2 increases glutamate uptake through overexpression of GLT1 and EAAC1 glutamate transporter subtypes in rat frontal cerebral cortex.

Prenatal exposure to the CB1 receptor agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)-pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone) mesylate (WIN) at a daily dose of 0.5 mg/kg, and Delta9-tetrahydrocannabinol (Delta9-THC) at a daily dose of 5 mg/kg, reduced dialysate glutamate levels in frontal cerebral cortex of adolescent offspring (40-day-old) with respect to those born from vehicle-treated mothers. WIN treatment induced a statistically significant enhancement of Vmaxl-[3H]glutamate uptake, whereas it did not modify glutamate Km, in frontal cerebral cortex synaptosomes of adolescent rats. Western blotting analysis, performed either in membrane proteins derived from homogenates and in proteins extracted from synaptosomes of frontal cerebral cortex, revealed that prenatal WIN exposure enhanced the expression of glutamate transporter 1 (GLT1) and excitatory amino acid carrier 1 (EAAC1). Moreover, immunocytochemical analyses of frontal cortex area revealed a more intense GLT1 and EAAC1 immunoreactivity (ir) distribution in the WIN-treated group. Collectively these results show that prenatal exposure to the cannabinoid CB1 receptor agonist WIN increases expression and functional activity of GLT1 and EAAC1 glutamate transporters (GluTs) associated to a decrease of cortical glutamate outflow, in adolescent rats. These findings may contribute to explain the mechanism underlying the cognitive impairment observed in the offspring of mothers who used marijuana during pregnancy.

The glutamate and chloride permeation pathways are colocalized in individual neuronal glutamate transporter subunits.

Glutamate transporters have a homotrimeric subunit structure with a large central water-filled cavity that extends partially into the plane of the lipid bilayer (Yernool et al., 2004). In addition to uptake of glutamate, the transporters also mediate a chloride conductance that is increased in the presence of substrate. Whether the chloride channel is located in the central pore of the trimer or within the individual subunits has been controversial. We find that coexpression of wild-type neuronal glutamate transporter EAAT3 subunits with subunits mutated at R447, a residue governing substrate selectivity (Bendahan et al., 2000), results in transport activity consistent with two distinct noninteracting populations of transporters, in agreement with previous work suggesting that each subunit operates independently to transport substrate (Awes et al., 2004; Grewer et al., 2005; Koch and Larsson, 2005). In wild-type homotrimeric transporters, the glutamate concentration dependence of the anion conductance and the kinetics of glutamate flux were isolated and measured, and the anion channel activation was fitted to analytical expressions corresponding to (1) a central pore gated by binding to one or more subunits and (2) a channel pore in each subunit. The data indicate that glutamate-binding sites, transport pathways, and chloride channels reside in individual subunits in a trimer and function independently.

The glutamate-activated anion conductance in excitatory amino acid transporters is gated independently by the individual subunits.

Excitatory amino acid transporters (EAATs) use sodium and potassium gradients to remove glutamate from the synapse and surrounding extracellular space, thereby sustaining efficient synaptic transmission and maintaining extracellular glutamate concentrations at subneurotoxic levels. In addition to sodium-driven glutamate uptake, EAATs also mediate a glutamate-activated chloride conductance via a channel-like mechanism. EAATs are trimeric proteins and are thought to comprise three identical subunits. Previous studies have shown that the sodium-driven uptake of glutamate occurs independently in each of the three subunits. In contrast, a recent study reports high Hill coefficients for the activation of EAAT anion currents by glutamate and suggests that the subunits function cooperatively in gating the chloride conductance. In the present work, we find that the Hill coefficient for the activation of the anion current by glutamate is approximately 1 in both EAAT3 and EAAT4. Furthermore, we also used fluorescent labeling and inactivation correlation on EAAT3 and EAAT4 to determine whether the glutamate-activated chloride conductance is gated independently or cooperatively by the transporters. We found that both glutamate uptake currents and glutamate-activated chloride currents are mediated independently by each subunit of an EAAT multimer. It has been suggested that EAAT subtypes with particularly large anion conductances can directly influence the excitability of presynaptic terminals in certain neurons. Thus, the finding that the anion conductance is gated independently, rather than cooperatively, is important because it significantly alters predictions of the influence that EAAT-mediated anion currents will have on synaptic transmission at low glutamate concentrations.