Arginine Transport Activity Research Articles

Estradiol augments while progesterone inhibits arginine transport in human endothelial cells through modulation of cationic amino acid transporter-1.

Decreased generation of nitric oxide (NO) by endothelial NO synthase (eNOS) characterizes endothelial dysfunction (ECD). Delivery of arginine to eNOS by cationic amino acid transporter-1 (CAT-1) was shown to modulate eNOS activity. We found in female rats, but not in males, that CAT-1 activity is preserved with age and in chronic renal failure, two experimental models of ECD. In contrast, during pregnancy CAT-1 is inhibited. We hypothesize that female sex hormones regulate arginine transport. Arginine uptake in human umbilical vein endothelial cells (HUVEC) was determined following incubation with either 17β-estradiol (E2) or progesterone. Exposure to E2 (50 and 100 nM) for 30 min resulted in a significant increase in arginine transport and reduction in phosphorylated CAT-1 (the inactive form) protein content. This was coupled with a decrease in phosphorylated MAPK/extracellular signal-regulated kinase (ERK) 1/2. Progesterone (1 and 100 pM for 30 min) attenuated arginine uptake and increased phosphorylated CAT-1, phosphorylated protein kinase Cα (PKCα), and phosphorylated ERK1/2 protein content. GO-6976 (PKCα inhibitor) prevented the progesterone-induced decrease in arginine transport. Coincubation with both progesterone and estrogen for 30 min resulted in attenuated arginine transport. While estradiol increases arginine transport and CAT-1 activity through modulation of constitutive signaling transduction pathways involving ERK, progesterone inhibits arginine transport and CAT-1 via both PKCα and ERK1/2 phosphorylation, an effect that predominates over estradiol.

Cationic amino acid transport activity and detection of B‐defensin‐1 in canine lens epithelial cells

AbstractPurpose The aim of this study is to investigate the cationic amino acid transport activity in canine lens epithelial cells line (cdLEC).B‐defensins are small cationic peptides possessing a broad range of antimicrobial and physiological activities. The mucosal physical barrier is an important component protecting luminal surfaces from bacteria. Expression of B‐defensins was also examined.Methods Na‐independence of arginine transport activity was measured, and N‐ethylmaleimide (NEM)‐sensitive uptake of arginine was investigated. cDNA sequence of cationic amino acid transporter 1 (CAT1) was determined based on human and mouse CAT1 sequences. RT‐PCR analysis of B‐defensins was carried out.Results NEM‐sensitive component of arginine uptake was detected. cDNA sequence of CAT1 was determined from the cdLEC. The sequence was 2589 bp long and was predicted to encode the 629 amino acid polypeptides. The deduced amino acid sequence showed similarities of 92.1% and 88.6% to those of human and mouse, respectively. Western blot analysis indicated the single band at 70 kDa in membrane protein sample of cdLEC. RT‐PCR analysis confirmed that CAT1 was ubiquitously detected in all tissues examined. Among the B‐defensins, B‐defensin‐1 but not ‐2 and ‐3 were observed in cdLECs and primary culture of lens epithelial cells.Conclusion Lens epithelial cells possess CAT1, and CAT1 may provide substrates for synthesis of B‐defensin‐1.

Arginine transport in human monocytic leukemia THP-1 cells during macrophage differentiation

L-arginine metabolism in myeloid cells plays a central role in the processes of macrophage activation and in the regulation of immune responses. In this study, we investigated arginine transport activity and the expression of the related transporter genes during the differentiation of monocytes to macrophages. We show here that the induction of THP-1 monocyte differentiation by PMA markedly increases the expression of SLC7A7 mRNA and of y(+)LAT1 protein and consequently, the activity of system y(+)L-mediated arginine transport. Conversely, the activity of system y(+) decreases during macrophage differentiation as a result of a reduction in CAT1 protein expression. The PMA-induced, macrophage-differentiated phenotype and the increased activity of system y(+)L through the induction of SLC7A7 gene are mediated by the specific activation of PKCβ. SLC7A7 gene silencing causes a significant reduction of system y(+)L activity and a subsequent, marked increase of arginine and lysine cell content, thus suggesting that in macrophagic cells, system y(+)L activity is mainly directed outwardly. Differentiating agents other than PMA, i.e., VD3 and ATRA, are equally effective in the stimulation of system y(+)L transport activity through the increased expression of SLC7A7 mRNA and y(+)LAT1 protein. Moreover, we found that also during differentiation of human monocytes from peripheral blood SLC7A7 mRNA and system y(+)L activity are increased. These findings point to SLC7A7 gene as a marker of macrophage differentiation.

Arginine Homeostasis and Transport in the Human Pathogen Leishmania donovani

Arginine is an essential amino acid for the human pathogen Leishmania but not to its host. Thus, the mechanism by which this protozoan parasite regulates cellular homeostasis of arginine is critical for its survival and virulence. In a previous study, we cloned and functionally characterized a high affinity arginine-specific transporter, LdAAP3, from Leishmania donovani. In this investigation, we have characterized the relationship between arginine transport via LdAAP3 and amino acid availability. Starving promastigotes for amino acids decreased the cellular level of most amino acids including arginine but also increased the abundance of both LdAAP3 mRNA and protein and up-regulated arginine transport activity. Genetic obliteration of the polyamine biosynthesis pathway for which arginine is the sole precursor caused a significant decrease in the rate of arginine transport. Cumulatively, we established that LdAAP3 expression and activity changed whenever the cellular level of arginine changed. Our findings have led to the hypothesis that L. donovani promastigotes have a signaling pathway that senses cellular concentrations of arginine and subsequently activates a mechanism that regulates LdAAP3 expression and activity. Interestingly, this response of LdAAP3 to amino acid availability in L. donovani is identical to that of the mammalian cation amino acid transporter 1. Thus, we conjecture that Leishmania mimics the host response to amino acid availability to improve virulence.

Regulation of Amino Acid Arginine Transport by Lipopolysaccharide and Nitric Oxide in Intestinal Epithelial IEC-6 Cells

As a precursor for nitric oxide (NO) synthesis and an immune-enhancing nutrient, amino acid L-arginine plays a critical role in maintaining intestine mucosal integrity and immune functions in sepsis. However, the relationship between intestinal arginine transport and NO synthesis in sepsis remains unclear. In the present study, we investigated the effects of lipopolysaccharide (LPS) and NO on the arginine transport in cultured rat intestinal epithelial IEC-6 cell. Near-confluent IEC-6 cells were incubated with LPS (0-50 microg/ml) in serum-free Dulbecco's modified Eagles's medium, in the presence and absence of the NO donor sodium nitroprusside (SNP, 0-500 micromol/L) and the inducible nitric oxide synthase (iNOS) inhibitor N-omega-nitro-L-arginine (NNA, 0-1000 micromol/L) for various periods of time (0-48 hours). Arginine transport activity, arginine transporter CAT1 mRNA and protein levels were measured with transport assay, Northern blot analysis, and Western blot analysis, respectively. LPS increased arginine transport activity in a time- and dose-dependent fashion. Prolonged incubation of LPS (24 hours, 25 microg/ml) resulted in a 3-fold increase of arginine transport activity (control: 28 +/- 5; LPS: 92 +/- 20 pmol/mg/min, P < 0.05), with the System y(+) as the predominant arginine transport system, and a 2-fold increase of System y(+)CAT1 mRNA and transporter protein levels (P < 0.05). LPS increased the arginine transport System y(+) maximal velocity (V(max), control: 1484 +/- 180; LPS: 2800 +/- 230 pmol/mg/min, P < 0.05) without affecting the transport affinity (K(m), control: 76 +/- 8; LPS: 84 +/- 14 micromol/L, p = NS). The LPS-induced arginine transport activity was blocked by sodium nitroprusside (SNP) (control: 25 +/- 6; LPS: 97 +/- 26 *; SNP: 22 +/- 0.4(+); LPS+SNP: 33 +/- 10.3(+) pmole/mg/min, *P < 0.01 and (+)p = NS, compared with control). In contrary, the LPS-induced arginine transport activity was further augmented by NNA (control: 18 +/- 3.2; LPS: 59 +/- 2.7 *; NNA: 26.3 +/- 5.8; LPS + NNA: 127 +/- 18(+) pmol/mg/min; *P < 0.01 compared with control and (+)P < 0.01 compared with control or LPS). LPS-stimulates arginine transport activity in IEC-6 cells via a mechanism that involves increase of transport System y(+) mRNA levels and transporter protein levels. The LPS-stimulated arginine transport activity is regulated by the availability of nitric oxide.

Protein kinase C activation mediates acidosis down-regulation of intestinal arginine absorption in proliferating rat crypt cells

Introduction. The small intestinal undifferentiated crypt cells and differentiated villous cells serve different physiological functions in response to various stimuli. L-Arginine is the exclusive precursor for nitric oxide (NO) biosynthesis and an important nutrient for intestinal integrity during stress states. The purpose of this in vitro study was to investigate the poorly understood intracellular signaling pathways involved in the acidosis regulation of intestinal membrane arginine absorption in the undifferentiated rat intestinal crypt IEC-6 cells. Methods. IEC-6 cells were grown to subconfluence and incubated in growth media of various pHs (pH 6.6 to pH 7.4, adjusted with 1 M HCl and/or NaHCO 3), ±PKC inhibitor Chelerythrine Cl (CHEL, 0–6.6 μM), Phosphatidylinositide-3 kinase (PI-3 kinase) inhibitors wortmannin (0–10 μM), and LY 294004 (LY, 0–10 μM). 3H-arginine (5 μm) transport activity, arginine transporter MCAT1 mRNA levels, cellular protein kinase C (PKC) levels were measured. Data are means ± SD and analyzed by ANOVA with P < 0.05. Results. Desired extracellular pH (pH 6.6 to pH 7.4) was achieved in 6 h and remained stable. Chronic acidosis (pH 6.6, <48 h) resulted in a 48% decrease of arginine transport activity and a 60% increase in cellular phospho-PKC-pan levels, with significant increases in phospho-PKC-δ and phospho-PKC-μ isoforms but not the phospho-PKC-αβ, phospho-PKC-ε, or phospho-PKC-ζ isoforms. Protein kinase C inhibitor chelerythrine Cl and PI-3 kinase inhibitors wormannin and LY 294004 individually blocked the acidosis-mediated reduction in arginine transport activity and MCAT1 mRNA levels. Conclusions. Protein kinase C activation is a key intracellular regulator in the signal transduction cascade involved in this acidosis-mediated reduction in membrane arginine transport activity in the undifferentiated rat intestinal epithelial cells. TABLE—ABSTRACT 9 Control + CHEL + Wormannin + LY Control 0.12 ± 0.03 0.11 ± 0.02 0.11 ± 0.03 0.09 0.04 Acidosis 0.062 ± 0.02 ∗ 0.13 ± 0.03 0.09 ± 0.01 0.10 ± 0.01 Unit: nmole/mg/min. ∗ P < 0.05

Mitogen-Activated Protein Kinases (MAPK) mediates Epidermal Growth Factor (EGF) up regulation of arginine transport in cultured intestinal epithelia

Introduction. Intestinal luminal growth factor EGF plays a key role in regulating intestinal epithelial growth and differentiation as well as amino acid absorption. However, little is known about the intracellular regulatory mechanisms involved. The purpose of this in vitro study was to delineate the intracellular signaling pathways involved in the EGF stimulation of arginine transport in cultured intestinal epithelial cells. Methods. IEC-6 cells were grown to sub-confluence and incubated in EGF (0–200 ng/ml), ± inhibitors of MAPK ERK1 (PD 98059, 0–50 μM) or MAPK p38 (SB 203580, 0–10 μM). 3H-arginine (5 μm to 10 mM) transport activity, arginine transporter MCAT1 mRNA, and MAPK levels were measured. Data are means ± SD and analyzed by ANOVA with P < 0.05. Results. EGF stimulated arginine transport activity in a dose- and time-dependent fashion. Prolonged EGF exposure (100 ng/ml, 48 h) resulted in a 48% increase of arginine transport and a 2.5-fold increase in transporter MCAT1 mRNA levels. The transport activity increase was the result of an increase of maximal transport velocity of both Systems y + (V max 40 ± 7 control versus 74 ± 13 EGF, nmol/mg/min, P < 0.05) and L (V max 9.8 ± 1.2 control versus 17 ± 2.3 EGF, P < 0.05). The transport affinity of both systems was not affected ( P = NS). EGF increased the phopspho-MER1, phospho-p44/42, and phospho-p38 levels, the active forms of MAPK, without affecting the total MAPK levels. PD 98059 or SB 203580 individually attenuated the EGF-induced arginine transport activity and transporter mRNA levels. Conclusion. EGF stimulates arginine transport activity and transporter gene expression in IEC-6 cells via an intracellular MAPK signaling cascade. TABLE—ABSTRACT P14 Treatment Control PD 98059 SB 203580 Control 0.213 ± 0.11 0.238 ± 0.03 0.225 ± 0.05 EGF 0.309 ± 0.03 ∗ 0.256 ± 0.02 ∗∗ 0.245 ± 0.03 ∗∗ Note. Unit: nmole/mg/min; ∗ p < 0.05 versus control; ∗∗ p < 0.05 versus EGF alone.

Arginine Transport in Catabolic Disease States

Arginine appears to be a semiessential amino acid in humans during critical illness. Catabolic disease states such as sepsis, injury, and cancer cause an increase in arginine utilization, which exceeds body production, leading to arginine depletion. This is aggravated by the reduced nutrient intake that is associated with critical illness. Arginine depletion may have negative consequences on tissue function under these circumstances. Nutritional regimens containing arginine have been shown to improve nitrogen balance and lymphocyte function, and stimulate arginine transport in the liver. We have studied the effects of stress mediators on arginine transport in vascular endothelium, liver, and gut epithelium. In vascular endothelium, endotoxin stimulates arginine uptake, an effect that is mediated by the cytokine tumor necrosis factor-alpha (TNF-alpha) and by the cyclo-oxygenase pathway. This TNF-alpha stimulation involves the activation of intracellular protein kinase C (PKC). A significant increase in hepatic arginine transport activity also occurs following burn injury and in rats with progressive malignant disease. Surgical removal of the growing tumor results in a normalization of the accelerated hepatic arginine transport within days. Chronic metabolic acidosis and sepsis individually augment intestinal arginine transport in rats and Caco-2 cell culture. PKC and mitogen-activated protein kinases are involved in mediating the sepsis/acidosis stimulation of arginine transport. Understanding the regulation of plasma membrane arginine transport will enhance our knowledge of nutrition and metabolism in seriously ill patients and may lead to the design of improved nutritional support formulas.

Metabolic acidosis stimulates intestinal arginine absorption

Introduction: L-Arginine is the exclusive precursor for nitric oxide (NO) biosynthesis and an essential nutrient for intestinal integrity during stress states. However, the effect of extracellular pH on intestinal arginine uptake is poorly understood. The purpose of this in vitro study was to investigate the regulation of intestinal arginine transport during acidosis. Methods:3H-L-arginine transport activity was measured in intestinal epithelial Caco-2 cells at various ambient pHs (pH 6.6–7.6) ± actinomycin-D (1 μM), cycloheximide (20 μM), phosphatidylinositide-3 kinase (PI-3) inhibitor wortmannin (1 uM), and the NO synthetase inhibitor N-ω-Nitro-L-Arginine (L-NA, 100 μM). Data were analyzed by ANOVA. Results: Decreasing the extracellular pH stimulated arginine transport activity in a time and pH-dependent fashion. Chronic acidosis (pH 6.6 for 48 hours) resulted in a 4-fold increase in arginine transport activity (0.94 ± 0.10 acidosis vs. 0.23 ± 0.05 control, nmole/mg/min, p < 0.01) and a 3-fold increase of arginine transporter mCAT-1 mRNA levels (p < 0.01). This acidosis-induced increase in arginine transport activity was due to a stimulation of transporter maximal transport capacity (Vmax 1.53 ± 0.08 acidosis vs. 0.56 ± 0.05 control, nmole/mg /min, p < 0.01) rather than a change in transporter affinity (Km = 54 ± 10 acidosis vs. 43 ± 6 control, μM arginine, p = NS). This acidosis-stimulated arginine transport activity was individually blocked by actinomycin-D, cycloheximide, and L-NA. The acidosis-induced arginine transport activity was further stimulated by wortmannin (1.4 ± 0.25 acidosis + wortmannin vs. 0.94 ± 0.10 acidosis vs. 0.21 ± 0.03 wortmannin vs. 0.23 ± 0.05 control, nmole/mg /min). Conclusion: Acidosis stimulates arginine transport in Caco-2 cells via a mechanism that leads to transcription of the gene and translation of arginine transporter. Phosphatidylinositide-3 kinase is a key intracellular regulator involved in this signal transduction cascade. The nitric oxide synthetase activity plays a pivotal role in this stimulation. An increased availability of arginine to cells during acidosis provides substrate for NO biosynthesis and may help in maintaining cellular integrity.

Activation of Intestinal Arginine Transport by Protein Kinase C Is Mediated by Mitogen-Activated Protein Kinases

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 μmol/L), and the MEK1 inhibitor PD 98059 (0 to 50 μmol/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. ( J Gastrointest Surg 2002;6:876–882.)

Specific Reversible Stimulation of System y +l-Arginine Transport Activity in Human Intestinal Cells

l-Arginine, which is intimately involved in cellular immune functions and nitric oxide biology, is transported by intestinal cells largely via transport System y +. The gut epithelium is exposed to various luminal amino acids at any given time, and therefore the purpose of this study was to study the regulation of luminal arginine transport by other amino acids. System y + l-arginine transport activity was measured in Caco-2 monolayers exposed to various amino acids. l-arginine and/or other System y + substrates specifically upregulated System y + transport activity twofold after 1 hour, with a response noted as early as 5 minutes. Non–System y + substrates did not affect l-arginine absorption. Kinetic analysis indicated that l-arginine exposure increased both System y + K m and V max. Neither cycloheximide nor actinomycin affected this stimulation, indicating that the regulation did not involve transcription or translation. The System y + substrate activation effect was reversible. l-arginine transport activity returned to baseline within 3 hours when cells were reincubated in amino acid–free media. These data indicate that System y + arginine transport activity is rapidly and reversibly activated by System y + substrates via a mechanism consistent with transmembrane stimulation. These findings identify a mechanism by which luminal nutrients regulate arginine uptake by the gut. ( J Gastrointest Surg 2002;6:379–386.)

Specific reversible stimulation of system y+ arginine transport activity in human intestinal cells

Specific reversible stimulation of system y+ arginine transport activity in human intestinal cells

Specific reversible stimulation of system y+ arginine transport activity in human intestinal cells

Specific reversible stimulation of system y+ arginine transport activity in human intestinal cells

Cationic amino acid transporter gene expression in cultured vascular smooth muscle cells and in rats.

Immunostimulants trigger vascular smooth muscle cells (VSMC) to express the inducible isoform of NO synthase (iNOS) and increased arginine transport activity. Although arginine transport in VSMC is considered to be mediated via the y+ system, we show here that rat VSMC in culture express the cat-1 gene transcript as well as an alternatively spliced transcript of the cat-2 gene. An RT-PCR cloning sequence strategy was used to identify a 141-base nucleotide sequence encoding the low-affinity domain of alternatively spliced CAT-2A and a 138-base nucleotide sequence encoding the high-affinity domain of CAT-2B in VSMC activated with lipopolysaccharide (LPS) in combination with interferon-gamma (IFN). With this sequence as a probe, Northern analyses showed that CAT-1 mRNA and CAT-2B mRNA are constitutively present in VSMC, and the expression of both mRNAs was rapidly stimulated by treatment with LPS-IFN, peaked within 4 h, and decayed to basal levels within 6 h after LPS-IFN. CAT-2A mRNA was not detectable in unstimulated or stimulated VSMC. Arginine transporter activity significantly increased 4-10 h after LPS-IFN. iNOS activity was reduced to almost zero in the absence of extracellular arginine uptake via system y+. Induction of arginine transport seems to be a prerequisite to the enhanced synthesis of NO in VSMC. Moreover, this work demonstrates tissue expression of CAT mRNAs with use of a model of LPS injection in rats. RT-PCR shows that the expression of CAT-1 and CAT-2B mRNA in the lung, heart, and kidney is increased by LPS administration to rats, whereas CAT-2A mRNA is abundantly expressed in the liver independent of LPS treatment. These findings suggest that together CAT-1 and CAT-2B play an important role in providing substrate for high-output NO synthesis in vitro as well as in vivo and implicate a coordinated regulation of intracellular iNOS enzyme activity with membrane arginine transport.

Adaptive Regulation of the Cationic Amino Acid Transporter-1 (Cat-1) in Fao Cells

The regulation of the high affinity cationic amino acid transporter Cat-1 in Fao rat hepatoma cells by amino acid availability has been studied. Cat-1 mRNA level increased (3-fold) in 4 h in response to amino acid starvation and remained high for at least 24 h. This induction was independent of the presence of serum in the media and transcription and protein synthesis were required for induction to occur. When Fao cells were shifted from amino acid-depleted media to amino acid-fed media, the levels of the induced cat-1 mRNA returned to the basal level. In amino acid-fed cells, accumulation of cat-1 mRNA was dependent on protein synthesis, indicating that a labile protein is required to sustain cat-1 mRNA level. No change in the transcription rate of the cat-1 gene during amino acid starvation was observed, indicating that cat-1 is regulated at a post-transcriptional step. System y+ mediated transport of arginine was reduced by 50% in 1 h and by 70% in 24 h after amino acid starvation. However, when 24-h amino acid-starved Fao cells were preloaded with 2 mM lysine or arginine for 1 h prior to the transport assays, arginine uptake was trans-stimulated by 5-fold. This stimulation was specific for cationic amino acids, since alanine, proline, or leucine had no effect. These data lead to the hypothesis that amino acid starvation results in an increased cat-1 mRNA level to support synthesis of additional Cat-1 protein. The following lines of evidence support the hypothesis: (i) the use of inhibitors of protein synthesis in starved cells inhibits the trans-zero transport of arginine; (ii) cells starved for 1-24 h exhibited an increase of trans-stimulated arginine transport activity for the first 6 h and had no loss of activity at 24 h, suggesting that constant replenishment of the transporter protein occurs; (iii) immunofluorescent staining of 24-h fed and starved cells for cat-1 showed similar cell surface distribution; (iv) new protein synthesis is not required for trans-stimulation of arginine transport upon refeeding of 24-h starved cells. We conclude that the increased level of cat-1 mRNA in response to amino acid starvation support the synthesis of Cat-1 protein during starvation and increased amino acid transport upon substrate presentation. Therefore, the cat-1 mRNA content is regulated by a derepression/repression mechanism in response to amino acid availability. We propose that the amino acid-signal transduction pathway consists of a series of steps which include the post-transcriptional regulation of amino acid transporter genes.

Relevance of the arginine transport activity to the nitric oxide synthesis in mouse peritoneal macrophages stimulated with bacterial lipopolysaccharide.

Transport of arginine and production of nitrite have been investigated in mouse peritoneal macrophages stimulated with bacterial lipopolysaccharide (LPS). The arginine transport activity was induced by LPS at very low concentration (maximally induced at 1 ng/ml), whereas much higher concentration of LPS was required for the induction of nitrite production. Arginine was more concentrated in the cells when its transport activity was induced. Lysine, which is a competitive inhibitor of the transport of arginine, neutralized the concentrative effect of the induced transport activity and thus inhibited the nitrite production. Induction of the arginine transport activity seems to be prerequisite to the enhanced synthesis of nitric oxide in activated macrophages.

Lipopolysaccharide and tumor necrosis factor stimulate lung microvascular arginine uptake, a response attenuated by dexamethasone.

Lipopolysaccharide (LPS), tumor necrosis factor-alpha, (TNF), and glucocorticoids can modulate endothelial nitric oxide (NO) production. L-Arginine is the exclusive precursor for NO biosynthesis, suggesting that NO generation and arginine transport are intimately linked. To further study this relationship, we examined the effects of LPS, TNF, and dexamethasone (DEX) on arginine uptake by rat lung microvascular endothelial cells. The transport of radiolabeled arginine was assayed in confluent cells grown in 24-well plates. The bulk (> 90%) of arginine transport was mediated by the Na(+)-independent carriers System y+ and System b0,+. Arginine transport was stimulated independently by LPS and TNF, a response first observed at 10 hours. Together, both agents exerted an additive effect on carrier-mediated uptake. The LPS- and TNF-induced increase in arginine transport activity was blocked by cycloheximide and actinomycin D, indicating the requirement for RNA and protein synthesis. The enhancement in transport activity was primarily due to an increase in Systems y+ maximal transport capacity (Vmax) with no change in transporter affinity and little change in System b0,+ activity. Treatment of cells with dexamethasone inhibited arginine transport activity in a time- and dose-dependent manner, an event that was abrogated by both actinomycin D and cycloheximide. The combination of DEX and LPS and TNF abrogated each other's antagonistic effects. These data indicate that LPS and TNF additively stimulate arginine transport in lung microvascular endothelial cells via a pathway that requires de novo protein synthesis (possibly of the transporter protein itself) and that this response is attenuated by DEX.

Protein kinase C-dependent regulation of l-arginine transport activity in Caco-2 intestinal cells

The regulation of plasma membrane l-arginine transport activity was investigated in differentiated and undifferentiated states of the human intestinal cell line, Caco-2. The sodium-independent, leucine-insensitive uptake of l-arginine measured in this study has been assigned by us previously to system y + in Caco-2 cells. Treatment of cells with serum-free media containing epidermal growth factor (EGF), transforming growth factor α (TGFα), or the protein kinase C (PKC) activator 12- O-tetradecanoylphorbol 13-acetate (TPA), stimulated system y + arginine transport activity in Caco-2 cells. Transport upregulation by these growth factors or by TPA was blocked by cycloheximide or the PKC inhibitor chelerythrine. Arginine uptake was diminished during the course of differentiation, attributable to a reduction in the transport system y + capacity ( V max) with no change in apparent affinity ( K m). TPA stimulated arginine uptake required at least 3 h of continual exposure, and increased the membrane's transport capacity ( V max) in both undifferentiated and differentiated cells. TPA elevated the diminished transport V max of differentiated cells TPA to the elevated V max value associated with undifferentiated cells. We conclude that upregulation of arginine transport is part of a pleiotropic response to EGF/TGFα, and that this involves PKC and de novo synthesis of polypeptides associated with system y + transport activity.

The amino acid transport system y+L induced in Xenopus laevis oocytes by human choriocarcinoma cell (JAR) mRNA is functionally related to the heavy chain of the 4F2 cell surface antigen.

Injection of mRNA isolated from human placental choriocarcinoma cells (JAR) into Xenopus laevis oocytes induced the transport of the neutral amino acid leucine as well as the transport of the cationic amino acid arginine. The induced transport of leucine was predominantly Na(+)-dependent, whereas that of arginine was Na(+)-independent. The ratio of transport activity for these amino acids in mRNA-injected oocytes versus water-injected oocytes was much greater if the transport activity was measured at pH 5.5 instead of at pH 7.5. Leucine transport in mRNA-injected oocytes was inhibited to a marked extent by arginine and lysine. The bicyclic amino acid BCH and the N-methyl amino acid MeAIB had no effect. Arginine transport in mRNA-injected oocytes was insensitive to N-ethylmaleimide and inhibited markedly by micromolar concentrations of leucine in the presence of Na+. The inhibitory potency of leucine was reduced severalfold in the absence of Na+. These results indicated that the arginine transport activity induced in Xenopus oocytes by JAR cell mRNA was due to an amino acid transport system which is identical with or highly similar to y+L and that this system was responsible for a major portion of leucine transport activity measured in these oocytes. Northern blot analysis showed that normal placenta and JAR cells do not possess detectable levels of mRNA transcripts for D2, a protein closely related to the function of another transport system, namely bo.+, which is also involved in the cellular uptake of neutral and cationic amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Normalization of Tumor-Induced Increases in Hepatic Amino Acid Transport After Surgical Resection

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. 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. 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. 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.