Faculty Bibliography

Glutamine is an essential nutrient for gut functions, but the regulation of its uptake by intestinal mucosal cells is poorly understood. Given the pivotal role of epidermal growth factor (EGF) in regulating gut metabolism, growth, and differentiation, this in vitro study was designed to investigate the intracellular signaling pathways involved in the regulation of EGF-mediated intestinal glutamine transport in intestinal epithelia. Continuous incubation with EGF (>30 hours, 100 ng/ml) stimulated glutamine transport activity across intestinal epithelial Caco-2 cell apical membrane. Exposure to EGF for 48 hours resulted in an increase in transport activity (50%) and glutamine transport system B gene ATB(0) mRNA levels (ninefold). EGF stimulated glutamine transport activity by increasing the glutamine transporter maximal velocity (V(max)) without altering the transporter apparent affinity (K(m)). Furthermore, EGF stimulated both intracellular protein kinase C and mitogen-activated protein kinase MEK1/2 activities. The EGF-stimulated glutamine transport activity was attenuated individually by the specific protein kinase C inhibitor chelerythrine chloride and the mitogen-activated protein kinase MEK1 inhibitor PD 98059. These data suggest that EGF activates glutamine transport activity across intestinal epithelial membrane via a signaling mechanism that involves activation of protein kinase C and the mitogen-activated protein kinase MEK1/2 cascade. EGF activates glutamine transport via alterations in transporter mRNA levels and the number of functional copies of transporter units.

During chronic metabolic acidosis, renal glutamine utilization increases markedly. We studied the expression of the system N1 (SN1) amino acid transporter in the kidney during chronic ammonium chloride acidosis in rats. Acidosis caused a 10-fold increase in whole kidney SN1 mRNA level and a 100-fold increase in the cortex. Acidosis increased Na(+)-dependent glutamine uptake into basolateral and brush-border membrane vesicles (BLMV and BBMV, respectively) isolated from rat cortex (BLMV, 219 +/- 66 control vs. 651 +/- 180 pmol. mg(-1). min(-1) acidosis; BBMV, 1,112 +/- 189 control vs. 1,652 +/- 148 pmol. mg(-1). min(-1) acidosis, both P < 0.05). Na(+)-independent uptake was unchanged by acidosis in BLMV and BBMV. The acidosis-induced increase in Na(+)-dependent glutamine uptake was eliminated by histidine, confirming transport by system N. SN1 protein was detected only in BLMV and BBMV from acidotic rats. After recovery from acidosis, SN1 mRNA and protein and Na(+)-dependent glutamine uptake activity rapidly returned to control levels. These data provide evidence that regulation of expression of the SN1 amino acid transporter is part of the renal homeostatic response to acid-base imbalance.

L-Arginine uptake by the small intestine can play a pivotal role in regulating nitric oxide synthesis and immune functions in catabolic states. We previously showed that protein kinase C (PKC) activation stimulates intestinal brush-border membrane arginine transport. However, the signaling pathways implicated in this activation have not been studied. The purpose of this study was to investigate the intracellular signal transduction pathways involved in the protein kinase C stimulation of arginine transport across the apical membrane of intestinal epithelial Caco-2 cells. [3H]-L-arginine transport activity, Northern blot analysis of mRNA levels of the intestinal arginine transporter CAT1, and Western blot analysis of the mitogen-activated protein (MAP) kinases phospho-p44/42 activity and phospho-MEK1/2 were measured in cultured Caco-2 cells treated with phorbol ester (phorbol 12-myristate 13-acetate, TPA; 0 to 0.5 micromol/L), and the MEK1 inhibitor PD 98059 (0 to 50 micromol/L). Phorbol ester stimulated intestinal arginine transport activity. Arginine transporter gene CAT1 mRNA, phospho-p44/42, and phospho-MEK1/2 levels were stimulated in phorbol ester-treated cells, compared with the control group. Phorbol ester stimulation of arginine transport activity and transporter CAT1 mRNA levels was blocked by PD 98059. These data suggest that phorbol ester stimulates arginine transport in Caco-2 cells via signaling pathways that lead to increased transcription and/or stabilization of CAT1 mRNA. Protein kinase C and MAP kinases MEK1/2 and p44/42 are key intracellular regulators involved in this signal transduction cascade.

BACKGROUND:Neutral dietary amino acids, such as alanine, are transported across the gut lumen by both Na(+)-dependent (System B) and Na(+)-independent (System L) carriers, but the nature of the acute phase of substrate-induced uptake is unknown. This study examined the effects of acute amino acid substrate exposure on the rapid modulation of apical membrane alanine transport in cultured human intestinal cells. METHODS:System B and System L transport activity kinetics, as well as ATB(0) mRNA levels, were measured in confluent Caco-2 monolayers treated with various metabolic agents during short-term and extended time periods. RESULTS:Depleting the incubation medium of alanine attenuated both System B and System L uptake activities within 30 mins, with a complete return to baseline values within 3 h. Extracellular alanine added to depleted Caco-2 cells rapidly (within 5 min) increased alanine transport activities. Kinetic analysis showed that acute alanine exposure increased both K(m) and V(max) of each transport system, indicative of a trans-stimulation effect. Augmenting intracellular alanine levels using the cytosolic alanine aminotransferase inhibitor, aminooxyacetic acid, increased alanine uptake activity. Acute exposure to other substrates of Systems B and L also increased the uptake of alanine, while nonsubstrates did not affect alanine uptake. Cycloheximide or actinomycin did not affect substrate acute activation of System B, and the steady-state level of ATB(0) mRNA was not altered by amino acid exposure. CONCLUSION/CONCLUSIONS:Increasing alanine availability to intestinal cells, by either exogenous substrate exposure or inhibition of intracellular catabolism, acutely and reversibly increases apical membrane alanine transport activity via a posttranslation trans-stimulation mechanism.