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W W Souba

Publications and source records attributed to W W Souba.

At least 19 recordsLinked to original sources
PubMed1995

Enhanced hepatic amino acid transport in tumor-bearing rats is partially blocked by antibody to tumor necrosis factor.

The liver of the host with cancer exhibits an enhanced requirement for amino acids to support tumor-induced increases in hepatic protein synthesis and gluconeogenesis. To address the mechanism by which the liver ensures adequate delivery of these substrates for intracellular utilization during cancer, we studied the activities of several amino acid transporters in hepatic plasma membrane vesicles prepared from rats implanted with a rapidly growing s.c. fibrosarcoma. The presence of the tumor resulted in a generalized stimulation of concentrative (Na(+)-dependent) glucogenic (small neutral) amino acid uptake via System A (3.4-fold), System N (2.3-fold), and System ASC (1.7-fold), as well as in the facilitative (Na(+)-independent) uptake of arginine via System y+ (1.7-fold). Kinetic analysis revealed that the tumor-induced enhancement of transport activity was due to increases in the maximum transport velocity (Vmax), whereas transporter substrate affinities (Km) did not change significantly. Administration of antibody to tumor necrosis factor-alpha to tumor-bearing rats attenuated the increase in hepatic amino acid transport activity by 60-100%. Treatment of nontumor-bearing control rats with tumor necrosis factor-alpha mAb did not alter basal transport activity. The results from these studies suggest that the tumor elicits a generalized increase in hepatic plasma membrane amino acid transport activity via a pathway that involves the cytokine tumor necrosis factor.

Amino Acids↗
PubMed1995

Detection of a functional promoter/enhancer in an intron-less human gene encoding a glutamine synthetase-like enzyme.

A human genomic clone, psi GS, containing an intron-less glutamine synthetase (GS)-encoding pseudogene, was isolated by screening a human library. A sequence of 3004 bp, containing the GS coding region and both the 5' and 3' flanking sequences, was identified that exhibits all the characteristics of a processed pseudogene. The coding region shows 93% identity with the human GS cDNA (hGS) sequence and contains two frame-shifts and two termination codons. The coding sequence is flanked by a 9-bp AT repeat and a putative polyadenylation site, AATAAA, at the 3' end. Primer extension analysis and S1 nuclease mapping showed a transcription start point (tsp) 62 bp upstream from the start codon indicating a shorter untranslated region than hGS. Transfection of HeLa cells with cat constructs containing portions of the 5' flanking sequence showed the presence of a functional promoter/enhancer within 200 bp of the tsp, independent of its orientation.

Amino Acid Sequence↗
PubMed1995

Protein kinase C activation inhibits glutamate transport by endothelial cells.

The role of protein kinase C (PKC) in regulating endothelial cell glutamate transport was investigated. Glutamate transport studies were performed in confluent human umbilical vein endothelial cells which were treated with the phorbol ester 12-myristate 13-acetate (TPA, 0-1000 nM), a compound which directly activates PKC. TPA inhibited Na(+)-independent System xAG- glutamate transport by 70% but only slightly reduced Na(+)-dependent activity. The TPA-mediated reduction in transport activity was dose-dependent, beginning at 5 min and lasting for at least 24 hr. TPA inhibition of glutamate transport had two distinctive phases: an acute phase (< 1 hr, not affected by either cycloheximide or actinomycin D) in which TPA decreased System xAG- glutamate transporter affinity (TPA Km = 522 +/- 25 microM vs control Km = 329 +/- 85 microM, P < 0.01) but did not alter transporter capacity (TPA Vmax = 4426 +/- 230 pmole/mg/min vs control Vmax = 4535 +/- 750 pmole/mg/min, P = NS) and a chronic phase (4-24 hr) in which TPA inhibition of glutamate transport was due to a reduced transporter capacity (Vmax = 2895 +/- 570 pmole/mg/min) without altering transporter affinity (Km = 370 +/- 60 microM glutamate) and was abrogated by cycloheximide or actinomycin D. The protein kinase C inhibitor chelerythrine chloride abrogated TPA's inhibition effect in both the acute and chronic phases. These data indicate that protein kinase C activation decreases glutamate transport in human umbilical vein endothelial cells via protein synthesis dependent and independent mechanisms.

Biological Transport↗
PubMed1995

Tumor necrosis factor stimulates system XAG- transport activity in human endothelium.

System xAG- is responsible for the carrier-mediated Na(+)-independent transport of anionic amino acids such as glutamate and aspartate across the plasma membrane of cells. In order to examine a possible role for cytokines in regulating System xAG- activity, the effect of TNF on [3H]glutamate transport in cultured human umbilical vein endothelial cells (HUVECs) was studied. Carrier-mediated glutamate uptake was accomplished by two high-affinity carriers, predominantly by a Na(+)-independent carrier (System xAG-, 75% of total glutamate uptake) and, to a lesser extent by a Na(+)-dependent carrier (System XAG-, 24% of total uptake). TNF treatment (10 ng/ml for 10 hr) resulted in an 80% increase in Na(+)-independent glutamate transport activity with no change in System XAG- activity. The TNF stimulatory effect was blocked by actinomycin D and cycloheximide. TNF treatment increased System xAG- glutamate transporter Vmax by 51% (control Vmax = 2359 +/- 345 pmole/mg protein/min vs TNF Vmax = 3569 +/- 436 pmole/mg protein/min, P < 0.01) without altering transporter affinity (control Km, 229 +/- 40 microM glutamate vs TNF Km = 224 +/- 60 microM glutamate, P = NS). The protein kinase C (PKC) inhibitor chelerythrine chloride had no effect on the TNF-stimulated glutamate transport, indicating that the augmented glutamate transport was not mediated by PKC activation. These data indicate that the TNF-stimulated glutamate transport in HUVECs requires do novo protein synthesis, possibly of the System xAG- transporter protein itself. Accelerated glutamate transport provides a precursor for the biosynthesis of macromolecules and glutamine.

Biological Transport↗
PubMed1995

Attenuation of the endotoxin-stimulated increase in hepatic amino acid transport with a glucocorticoid receptor antagonist.

The role of the glucocorticoid hormones in mediating the accelerated hepatic amino acid transport that is characteristic of endotoxemia was investigated. To determine the role of these steroid hormones, rats that received endotoxin (LPS) were pretreated with the glucocorticoid receptor antagonist RU38486. The activities of the Na(+)-dependent amino acid transport systems A, ASC, and N and the Na(+)-independent systems L, y+, n, b0,+, and asc in hepatic plasma membrane vesicles were measured 4 hr after exposure to LPS. Endotoxin treatment resulted in time- and dose-dependent 5-fold (System A), 2.5-fold (System N), 2.6-fold (System ASC), and 2-fold (System y+ and b0,+) increases in transport activity attributable to an increase in carrier Vmax. The activities of L, asc, and n were unchanged by LPS administration. Pretreatment of endotoxemic animals with RU38486 attenuated the LPS-induced enhancement in transport activity by 20-60% by diminishing carrier Vmax, with no effect on transport Km. We conclude that the marked increase in hepatic amino acid transport activity that occurs during endotoxemia requires participation of the glucocorticoid hormones.

Amino Acids↗
PubMed1995

Glucocorticoids regulate rat glutamine synthetase expression in a tissue-specific manner.

During stress states, organismal glutamine production is augmented secondary to an increase in the activity of glutamine synthetase (GS) in the lung and skeletal muscle. Because glucocorticoids are key regulators of the metabolic response to stress, we undertook a survey of glucocorticoid induction of GS expression in rat organs in response to dexamethasone. Male adult rats were injected with glucocorticoid or vehicle and 4 hr later, 10 organs were assayed for GS messenger RNA and protein contents by Northern and Western blotting. We observed a 20-fold range of GS mRNA levels in organs of control animals. Blotting detected two GS RNA species of approximately 2.8- and 1.4-kb sizes in all tissue except testis, where an additional 2-kb RNA species was observed. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were also assayed and used as a normalization factor. An approximately 10-fold range of GAPDH mRNA levels was observed. Four hours after dexamethasone injection, a nearly a 5-fold increase in glutamine synthetase mRNA levels in lung and muscle, as well as an approximately 2-fold increase in heart were observed. Relative to GAPDH mRNA, a significant decrease in GS mRNA levels was observed in the liver. A wide range of glutamine synthetase protein contents were observed in rat organs. Comparison of Northern and Western blotting results revealed a dichotomy in the ratio of relative GS mRNA and protein level in rat organs, suggesting that tissue-specific posttranscriptional processes determine GS protein levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Characterization and growth factor stimulation of L-arginine transport in a human colon cancer cell line.

BACKGROUND: Epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) are potent mitogens that contribute to abnormal growth regulation in colon cancer. Growth factors have been shown to regulate transmembrane nutrient uptake as an adaptive response to support cellular proliferation. METHODS: The transport of L-arginine by the SW480 primary human colon adenocarcinoma cell line was characterized by assaying the uptake of [3H]L-arginine in the presence and absence of sodium. Kinetic studies were performed over a range of L-arginine concentrations to determine transport affinity (Km) and maximal transport velocity (Vmax). To further characterize the specific transporters, [3H]L-arginine uptake was measured in the presence of selected amino acids, hormones, and under conditions of varying external pH. To investigate the effects of EGF and TGF alpha, cells were incubated with increasing doses of growth factors (1, 10, 50 ng/ml) and L-arginine transport was measured at various time intervals (8, 12, 24 h). Proliferation was assessed by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay 3 days after growth factor stimulation. RESULTS: The majority of carrier-mediated L-arginine transport was via a sodium-independent process (65-70%), whereas the remainder was sodium-dependent (28-30%). Diffusion contributed a small amount to total L-arginine uptake (2%). Kinetic studies of arginine transport revealed a single high-affinity Na(+)-independent transporter with a Km = 55.8 +/- 5.8 microM and a Vmax = 710.6 +/- 87.3 pM/mg protein/30 s. Na(+)-independent arginine uptake was pH-insensitive and markedly inhibited by system y+ substrates L-homoarginine, L-lysine, and L-ornithine. A single Na(+)-dependent transporter with a Km = 19.8 +/- 2.3 microM and a Vmax = 159.1 +/- 8.9 pM/mg protein/30 s was identified. Na(+)-dependent arginine uptake was inhibited by system BO,+ substrates L-lysine, L-ornithine, L-leucine, L-cysteine, and L-glutamine, but not by 2-methylaminoisobutyric acid. In addition, Na(+)-dependent arginine uptake was pH- and hormone-insensitive. Incubation with EGF or TGF alpha had no effect on Na(+)-independent L-arginine uptake; however, Na(+)-dependent uptake was enhanced 60% by EGF (10 ng/ml, p < 0.05) and 100% by TGF alpha (10 ng/ml, p < 0.05), whereas cellular proliferation was increased 27% by EGF (10 ng/ml, p < 0.05) and 37% by TGF alpha (10 ng/ml, p < 0.01). CONCLUSIONS: L-arginine transport in the SW480 colon cancer cell line is principally mediated by the Na(+)-independent system y+ and to a lesser extent by the Na(+)-dependent system BO,+. Furthermore, EGF and TGF alpha preferentially stimulate L-arginine uptake via the Na(+)-dependent transporter, ostensibly to accommodate for the mitogenic stimulus.

Adenocarcinoma↗
PubMed1995

Dietary regulation of the hepatic system n glutamine transporter in tumor-bearing rats.

BACKGROUND: Hepatocytes possess a novel, plasma-membrane, sodium ion (Na+)-independent, glutamine transporter (system n), which functions to transport glutamine out of the cell into the blood. In the tumor-bearing rat, the activity of system n increases but its regulation is unknown. We hypothesized that the increase in system n that occurs in rats with cancer was related to a fall in the circulating glutamine concentration. METHODS: Ten male rats underwent flank implantation with a cube of methylcholanthrene-induced fibrosarcoma cells and 10 rats underwent a sham operation. After 9 days of standard diet, all rats were randomized to receive either a glutamine-enriched oral diet or an isonitrogenous diet without supplemental glutamine, for 1 week. Tumors and livers were harvested 16 days postimplantation. Arterial blood samples were obtained from all animals. Hepatic plasma membrane vesicles were prepared and the carrier-mediated, Na(+)-independent transport of glutamine was assayed. RESULTS: When compared to nontumor-bearing animals, tumor-bearing rats that were fed a control diet exhibited hypoglutaminemia and a 2.3-fold increase in the activity of system n. Glutamine dietary supplementation produced blood glutamine levels that were similar in both tumor-bearing and nontumor-bearing rats, apparently abrogating the increase in system n activity that was observed in tumor-bearing rats that were not fed supplemental glutamine. Tumor-bearing animals receiving supplemental glutamine had a decreased number of system n carriers (Vmax) in the hepatic plasma membrane compared to that of tumor-bearing animals receiving a control diet; this apparently abrogated the glutamine efflux rate. Glutamine feeding did not alter system n activity in nontumor-bearing controls. CONCLUSIONS: In the tumor-bearing animal model, system n is modulated by the circulating glutamine concentration. This is the first study that demonstrates the ability of specialized nutrition to "downregulate" transport activity in vivo. Provision of glutamine-enriched diets to the host with cancer may maintain hepatic glutamine levels and prevent host glutamine depletion.

Animals↗
PubMed1995

Cytokines regulate endotoxin stimulation of endothelial cell arginine transport.

BACKGROUND: Endotoxin (lipopolysaccharide) stimulates transmembrane L-arginine transport in pulmonary artery endothelial cells (PAECs). The proinflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) mediate many of the pathophysiologic effects of endotoxemia and sepsis. Endothelial cells secrete TNF and IL-1 in response to endotoxin. We hypothesize that lipopolysaccharide stimulation of plasma membrane L-arginine transport is mediated via an autocrine cytokine loop involving TNF and IL-1. METHODS: Confluent porcine PAECs were incubated with various concentrations of lipopolysaccharide, TNF, or IL-1, and arginine uptake was determined by assaying the uptake of 3H-L-arginine in the presence or absence of Na+ at different time points. PAECs were then incubated with lipopolysaccharide or saline solution after pretreatment with either anti-TNF antibody or IL-1-receptor antagonist, and transport was measured 12 hours later. RESULTS: Lipopolysaccharide, IL-1, and TNF all increased both Na+-dependent and Na+-independent carrier-mediated L-arginine transport in a fashion that was both time and dose dependent. Maximal increases in stimulated arginine uptake occurred 8 hours after exposure to the cytokines and 12 hours after exposure to lipopolysaccharide. Pretreatment of endothelial cells with anti-TNF antibody blocked lipopolysaccharide stimulation of both Na+-independent and Na+-dependent transport by 100% and 90%, respectively. In addition, IL-1-receptor antagonist inhibited lipopolysaccharide stimulation of both Na+-independent and Na+-dependent transport by 65% and 85%, respectively. CONCLUSIONS: The marked increase in carrier-mediated L-arginine transport activity produced by lipopolysaccharide, IL-1, and TNF may represent an adaptive response by the pulmonary endothelium to support arginine-dependent biosynthetic pathways during sepsis. Furthermore, lipopolysaccharide stimulation of arginine transport is mediated in part through an autocrine mechanism involving IL-1 and TNF.

Animals↗
PubMed1995

Strategies for success in academic surgery.

BACKGROUND: A growing concern among university surgeons is a perceived threat to the traditional academician role. Factors that have led to this concern include (1) changes in reimbursement and patient referral patterns, both of which have resulted in reduced income; (2) mounting external pressure from the departmental chairperson/dean to generate professional fees; (3) pervasive ambiguity in the criteria for promotion and tenure; (4) intensified competition for research dollars in recent years; and (5) a perception that fellow academicians view surgeons as operating room technicians incapable of laboratory research. These cogent issues have the means to place academic goals in jeopardy. Potentially they lead to dissatisfaction, unhappiness, and eventual departure from academia. A healthy solution to the problem includes departmental recognition and reward for whichever primary role/s the academic surgeon embraces--clinical, educational, research, or administrative. Clear chairperson-faculty communication on the issues of expectation and reward is a key ingredient in the solution. METHODS: A mail survey was conducted with chairpersons of surgical departments and academic surgeons addressing the issues of retention, promotion, and benefits. RESULTS: Results revealed significant differences in perceptions, suggesting the need for improved communication between faculty and chairpersons and a team approach to help maintain academic viability in the future. CONCLUSIONS: When the quadruple threat can be mutually described as a departmental rather than individual quality, the joys and rewards of academic surgery can be considerable for faculty and chairpersons alike.

Academic Medical Centers↗
PubMed1995

Molecular regulation of lung endothelial glutamine synthetase expression.

BACKGROUND: The lungs play a crucial role in maintaining amino acid homeostasis by exporting glutamine. Lung glutamine release is increased markedly in patients with sepsis, and in rat models injection of endotoxin causes up-regulation of glutamine synthetase (GS), the principal enzyme of glutamine synthesis. To investigate the molecular regulation of this response in the lung microvasculature we studied the effects of several hormones and cytokines that mediate the septic response on the expression of GS in rat microvascular pulmonary endothelial cells (MPECs). METHODS: MPECs were grown to confluence and incubated with the synthetic glucocorticoid dexamethasone, prostaglandins, cytokines, or activated complement C5a. Cellular lysates were prepared and total cellular RNA was extracted, hybridized with a GS complementary DNA derived probe, and normalized to reduced glyceraldehyde-phosphate dehydrogenase. GS protein content was determined by Western blotting with a GS antibody. RESULTS: Of the compounds tested, only dexamethasone caused a marked increase (tenfold or greater) of GS messenger RNA and protein levels in MPECs. Dexamethasone-induced accumulation of GS messenger RNA was rapid, dose-dependent, and maximal after 4 hours of exposure. GS protein levels were maximal after 8 hours and remained elevated for at least 48 hours. The dose of dexamethasone sufficient to induce 50% of maximal GS messenger RNA and protein level increase was approximately 10 nmol/L. The dexamethasone-induce increase of GS messenger RNA level was completely blocked by the glucocorticoid receptor antagonist RU38486 and by the transcriptional inhibitor actinomycin D but was not inhibited by the translational inhibitor cycloheximide. CONCLUSIONS: Glucocorticoids augment GS expression in rat lung microvascular endothelial cells in a manner consistent with a direct transcriptional response via glucocorticoid receptors. Other septic response mediators had minimal effect on GS expression. Induction of GS expression by adrenocorticoids is likely to contribute to the marked ability of the lungs to augment glutamine production during septic states.

Animals↗
PubMed1995

Normalization of tumor-induced increases in hepatic amino acid transport after surgical resection.

BACKGROUND: The liver of the host with cancer requires increased amounts of amino acids to support the synthesis of glucose and key defense proteins. To study the effect of the growing tumor on hepatic amino acid uptake, the authors measured hepatic transport activity in tumor-bearing rats and in rats at various times after tumor resection. METHODS: Fischer-344 rats were implanted subcutaneously with methylcholanthrene-induced fibrosarcoma cells (MCA sarcoma). When the tumors reached 10% of body weight, hepatic amino acid transport activity was assayed or the animals underwent surgical removal of the tumor. In animals that underwent tumor excision, livers were removed at 1, 3, or 5 days post-resection, and hepatic plasma membrane vesicles (HPMVs) were prepared. Nontumor-bearing pair-fed rats undergoing sham implantation or sham resection served as controls. System N (glutamine), System A (MeAIB), and System y+ (arginine) transport activity were assayed, which allowed the authors to compare differences in tumor-induced rates of transport and the influence of resection on transport activity. RESULTS: System A transport activity was unaltered by tumor growth. In contrast, the presence of the growing tumor increased arginine and glutamine uptake by the liver. Hepatic glutamine transport remained elevated for 5 days after tumor resection, although by postoperative day 5 there was a trend toward normalization. In contrast, arginine transport remained increased by twofold onpost-resection day 1 and had normalized by postoperative day 3. The enhanced arginine transport was a result of an increase in maximal transport velocity (Vmax) rather than a change in carrier affinity. CONCLUSIONS: Increases in hepatic amino acid transport normalize within several days of tumor resection, indicating a key role for the tumor in the induction of this response. The observation that hepatic glutamine transport activity remains augmented after tumor resection longer than any other transporter studied suggests a key role for this amino acid in overall hepatic nitrogen metabolism and may partially explain the persistent glutamine depletion that is characteristic of the tumor-bearing host.

Amino Acids↗
PubMed1995

TNF-stimulated arginine transport by human vascular endothelium requires activation of protein kinase C.

OBJECTIVE: The authors determined the endothelial arginine transport mechanism and the potential role of a tumor necrosis factor (TNF)-alpha-mediated signal transduction pathway involving protein kinase C (PKC) in regulating this transport in cultured endothelial cells. SUMMARY BACKGROUND DATA: The vascular endothelium metabolizes arginine to generate nitric oxide (NO), and an increase in NO production can be stimulated by several cytokines. The mechanism(s) responsible for the accelerated arginine transport are poorly understood. METHODS: Arginine transport was assayed in confluent human umbilical vein endothelial cells in the presence of TNF +/- the PKC inhibitor chelerythrine chloride. RESULTS: Carrier-mediated arginine transport was accomplished by two Na(+)-independent transporters, System y+ (80% of total transport) and System b0,+ (20% of transport). Tumor necrosis factor (0.1-2 ng/mL) increased System y(+)-mediated arginine transport in a time- and dose-dependent manner by augmenting System y+ transport maximal capacity (control Vmax = 1325 +/- 60 pmol/mg protein/minute vs. TNF Vmax = 3015 +/- 110 pmol/mg protein/minute, p < 0.01) without affecting transporter affinity (control Km = 30 +/- 1.4 microM vs. 34 +/- 1.3 microM arginine, p = NS). Stimulation was maximal at the 8-hour time point and was inhibited by both actinomycin D and cycloheximide. In addition, inhibition of PKC with chelerythrine abrogated the TNF-augmented arginine transport. Similarly, incubation of cells with the direct PKC activator TPA (phorbol ester 12-myristate 13-acetate) stimulated System y(+)-mediated arginine transport nearly fivefold, secondary to an increase in transporter Vmax (TPA Vmax = 5349 +/- 310 pmol/mg protein/minute, p < 0.001 vs. control), with no change in Km. This TPA-induced stimulation of arginine transport also was blocked by chelerythrine CI, actinomycin D, and cycloheximide. Incubation of TNF-stimulated cells with two NO synthase inhibitors did not reduce transport activity, suggesting that the arginine transporter and the NO synthase enzyme may, in part, be independently regulated.

Alkaloids↗
PubMed1995

Amino acid metabolism and the vascular endothelium: regulation and disease implications.

Amino acid metabolism by the vascular endothelium is a complex process that often begins with the carrier-mediated uptake of circulating amino acids into the endothelial cytoplasm. Amino acids are essential for maintaining intact endothelial functions, which include cell proliferation, regulation of blood flow and vascular tone, coagulation and fibrinolysis, and metabolism of a variety of macromolecules. The disturbances in endothelial amino acid transport and metabolism that occur during infection and inflammation are due, in part, to changes in substrate availability and to the local and/or systemic elaboration of specific mediators. An improved understanding of endothelial amino acid metabolism will not only provide new knowledge regarding disease mechanisms and regulation, but may also lead to new treatment strategies that may include the clinical use of specific nutritional formulas.

Amino Acid Sequence↗
PubMed1995

Hepatic uptake of glutamine and other amino acids during infection and inflammation.

Catabolic illness such as sepsis and injury induce profound changes in host amino acid metabolism, including increased hepatic amino acid uptake. Because many amino acid-dependent pathways such as gluconeogenesis and acute-phase protein synthesis are activated in the liver during severe infection, this review will focus on the control of hepatic plasma membrane amino acid transport by specific inflammatory mediators. We specifically review the role of cytokines, eicosanoids, and glucorticoids in this response. Collectively, these signaling molecules act in a concerted manner to exert local control of hepatic function including the stimulation of amino acid transport. In particular, we review the role of glutamine and its transport in the liver, as it occupies a unique role in interorgan ammonia metabolism during critical illness.

Acute-Phase Reaction↗
PubMed1995

Glutamine transport in isolated human hepatocytes and transformed liver cells.

The transport of L-glutamine was examined in isolated adult and fetal human hepatocytes as well as in the human hepatoma cell lines HepG2 and SK-Hep. In all cells studied, glutamine uptake was at least 85% Na(+)-dependent. Kinetic analysis of the Na(+)-dependent component indicated mediation by a single transporter in three human hepatocyte preparations and in SK-Hep cells, whereas two transporters appeared to be responsible for glutamine uptake in HepG2 cells and in hepatocytes from the liver of one male patient. Amino acid inhibition analysis showed primary mediation by System N in fetal and adult hepatocytes, whereas System ASC was principally responsible for glutamine uptake in transformed cells. Similar to the rat transporter, human System N was pH-sensitive, stereospecific, and responsive to treatment with steroid hormones. Although the human carrier was less tolerant of Li(+)- for Na+ substitution, glutamine transport in primary human hepatocytes was stimulated by treatment with hypotonic buffer (cell swelling), as reported in rat parenchymal cells. In contrast, glutamine transport in hepatoma cells was relatively insensitive to changes in extracellular pH and failed to show enhanced activity in response to hypoosmotic challenge. Collectively, the data suggest that markedly distinct plasma membrane transporters mediate the concentrative uptake of glutamine in normal and transformed human hepatocytes, and that the salient properties of System N have been largely conserved from rat to man.

Adult↗
PubMed1994

Glucocorticoids regulate intestinal glutamine synthetase gene expression in endotoxemia.

PURPOSE: Although glutamine is required to maintain gut mucosal metabolism and function, intestinal glutamine uptake from the gut lumen and from the bloodstream is decreased during sepsis. We hypothesized that endogenous mucosal glutamine biosynthesis is increased during endotoxemia, and we attempted to define the "stress" mediators that regulate the activity of small intestinal glutamine synthetase (GS), the principal enzyme of de novo glutamine biosynthesis in the gut. METHODS: Adult rats received Escherichia coli lipopolysaccharide (LPS) (7.5 mg/kg intraperitoneally), RU 38486 (a glucocorticoid antagonist; 10 mg/kg by gavage) 2 hours prior to LPS administration, antibody to tumor necrosis factor (TNF) (4 mg/kg intraperitoneally) prior to LPS administration, or ketorolac tromethamine (a prostaglandin synthesis inhibitor; 1 mg/kg intraperitoneally) followed by LPS administration. Mucosal GS activity was assayed 12 hours after LPS administration. In a separate set of studies, cultured intestinal mucosal cells (Caco-2) were exposed to LPS, interleukin 1 (IL-1), IL-6, TNF-alpha, interferon-gamma, prostaglandin E2, or dexamethasone. Twelve hours later, GS activity was assayed and messenger RNA was extracted. The GS transcripts were labeled with a GS complementary DNA probe radiolabeled with phosphorus 32, were quantitated by phosphoimaging, and were normalized to beta-actin. RESULTS: In vivo LPS treatment increased mucosal GS activity by 250%. Pretreatment with antibody to TNF or ketorolac did not inhibit the LPS-induced increase in mucosal GS, whereas pretreatment with RU 38486 attenuated the increase in gut GS activity by 60%. Lipopolysaccharide, IL-1, IL-6, TNF-alpha, gamma-interferon, and prostaglandin E2 did not increase GS activity in Caco-2 cells, whereas dexamethasone increased GS activity and messenger RNA 2.5-fold and threefold, respectively. These data indicate that cytokines and prostaglandins (prostaglandin E2) do not regulate mucosal GS expression during endotoxemia. Glucocorticoids, however, stimulate GS gene expression directly. CONCLUSIONS: This hormonally mediated response may support de novo mucosal GS during septic states when uptake of glutamine from the lumen and blood is decreased.

Animals↗