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Alveolar epithelium, besides exerting a key role in gas exchange and surfactant production, plays important functions in host defense and inflammation. Pathological conditions associated to alveolar dysfunction include Acute Respiratory Distress Syndrome (ARDS), asthma, chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). The use of predictive in vitro models of human alveolar epithelium is nowadays required for the study of disease mechanisms, as well as of pharmacokinetic parameters of pulmonary drugs delivery. Here, we employed a novel 3D model of human alveoli, namely EpiAlveolar™, consisting of primary alveolar epithelial cells, pulmonary endothelial cells and fibroblasts, that reflects properly the in vivo-like conditions. In EpiAlveolar™ we performed a characterization of Organic Cation Transporters (OCTs and OCTNs) expression and activity and we found that OCTN2, OCT1 and OCT3 are expressed on the basolateral membrane; instead, ATB0,+ transporter for cationic and neutral amino acids, which shares with OCTN2 the affinity for carnitine as substrate, is readily detectable and functional at the apical side. We also show that these transporters differentially interact with anticholinergic drugs. Overall, our findings reveal close similarities of EpiAlveolar™ with the tracheal/bronchial epithelium (EpiAirway™ model) and entrust this alveolar tissue as a potential tool for the screening of biopharmaceuticals molecules.

ATP-binding cassette (ABC) transporters are a large superfamily of membrane transporters that facilitate the translocation of different substrates. While ABC transporters are clearly expressed in various tumor cells where they can play a role in drug extrusion, the presence of these transporters in normal lung tissues is still controversial. Here, we performed an analysis of ABC transporters in EpiAlveolarTM, a recently developed model of human alveoli, by defining the expression and activity of MDR1, BCRP, and MRPs. Immortalized primary epithelial cells hAELVi (human alveolar epithelial lentivirus-immortalized cells) were employed for comparison. Our data underline a close homology between these two models, where none of the ABC transporters here studied are expressed on the apical membrane and only MRP1 is clearly detectable and functional at the basolateral side. According to these findings, we can conclude that other thus-far-unidentified transporter/s involved in drug efflux from alveolar epithelium deserve investigations.

Desmopressin (dDAVP) is the best characterized analogue of vasopressin, the endocrine regulator of water balance endowed with potent vasoconstrictive effects. Despite the use of dDAVP in clinical practice, ranging from the treatment of nephrogenic diabetes insipidus to bleeding disorders, much remains to be understood about the impact of the drug on endothelial phenotype. The aim of this study was, thus, to evaluate the effects of desmopressin on the viability and function of human pulmonary microvascular endothelial cells (HLMVECs). The results obtained demonstrate that the vasopressor had no cytotoxic effect on the endothelium; similarly, no sign of endothelial activation was induced by dDAVP, indicated by the lack of effect on the expression of inflammatory cytokines and adhesion molecules. Conversely, the drug significantly stimulated the production of nitric oxide (NO) and the expression of the inducible isoform of nitric oxide synthase, NOS2/iNOS. Since the intracellular level of cAMP also increased, we can hypothesize that NO release is consequent to the activation of the vasopressin receptor 2 (V2R)/guanylate cyclase (Gs)/cAMP axis. Given the multifaceted role of NOS2-deriving NO for many physio-pathological conditions, the meanings of these findings in HLMVECs appears intriguing and deserves to be further addressed.

y+LAT1 (encoded by SLC7A7), together with y+LAT2 (encoded by SLC7A6), is the alternative light subunits composing the heterodimeric transport system y+L for cationic and neutral amino acids. SLC7A7 mutations cause lysinuric protein intolerance (LPI), an inherited multisystem disease characterized by low plasma levels of arginine and lysine, protein‐rich food intolerance, failure to thrive, hepatosplenomegaly, osteoporosis, lung involvement, kidney failure, haematologic and immunological disorders. The reason for the heterogeneity of LPI symptoms is thus far only poorly understood. Here, we aimed to quantitatively compare the expression of SLC7A7 and SLC7A6 among different human cell types and evaluate y+LAT1 and y+LAT2 contribution to arginine transport. We demonstrate that system y+L‐mediated arginine transport is mainly accounted for by y+LAT1 in monocyte‐derived macrophages (MDM) and y+LAT2 in fibroblasts. The kinetic analysis of arginine transport indicates that y+LAT1 and y+LAT2 share a comparable affinity for the substrate. Differences have been highlighted in the expression of SLC7A6 and SLC7A7 mRNA among different cell models: while SLC7A6 is almost equally expressed, SLC7A7 is particularly abundant in MDM, intestinal Caco‐2 cells and human renal proximal tubular epithelial cells (HRPTEpC). The characterization of arginine uptake demonstrates that system y+L is operative in renal cells and in Caco‐2 where, at the basolateral side, it mediates arginine efflux in exchange with leucine plus sodium. These findings explain the defective absorption/reabsorption of arginine in LPI. Moreover, y+LAT1 is the prevailing transporter in MDM sustaining a pivotal role in the pathogenesis of immunological complications associated with the disease.

Background y+LAT1, encoded by SCL7A7, is the protein mutated in Lysinuric Protein Intolerance (LPI), a rare metabolic disease caused by a defective cationic amino acid (CAA, arginine, lysine, ornithine) transport at the basolateral membrane of intestinal and renal tubular cells. The disease is characterized by protein-rich food intolerance with secondary urea cycle disorder, but symptoms are heterogeneous with lung and immunological complications that are not explainable by the CAA transport defect. With the exception of the Finnish founder mutation (c.895-2A > T, LPIFin), LPI-causative mutations are heterogeneous and genotype-phenotype correlations have not been found. Here we addressed system y+L-mediated arginine uptake in monocytes from three LPI Italian patients and in lymphoblasts carrying the same mutations; in parallel, the genetic defects carried by the patients were reproduced as eGFP-tagged y+LAT1 mutants in transfected CHO cells to define the function and localization protein. Results System y+L activity is impaired in monocytes isolated from all LPI patients, and in CHO cells transfected with the three eGFP-y+LAT1 mutants, but not in lymphoblasts bearing the same mutations. The analysis of protein localization with confocal microscopy revealed that the eGFP-tagged mutants were retained inside the cytosol, with a pattern of expression quite heterogeneous among the mutants. Conclusions The three mutations studied of y+LAT1 transporter result in a defective arginine transport both in ex vivo (monocytes) and in vitro (CHO transfected cells) models, likely caused by the retention of the mutated proteins in the cytosol. The different effect of y+LAT1 mutation on arginine transport in monocytes and lymphoblasts is supposed to be due to the different expression of SLC7A7 mRNA in the two models, supporting the hypothesis that the impact of LPI defect largely depends on the relative abundance of LPI target gene in each cell type. Electronic supplementary material The online version of this article (10.1186/s13023-019-1028-2) contains supplementary material, which is available to authorized users.

The ATP-binding cassette (ABC) transporters P-glycoprotein (MDR1/ABCB1), multidrug resistance-associated protein 1 (MRP1/ABCC1), and breast cancer resistance protein (BCRP/ABCG2) play a crucial role in the translocation of a broad range of drugs; data about their expression and activity in lung tissue are controversial. Here, we address their expression, localization and function in EpiAirway™, a three-dimensional (3D)-model of human airways; Calu-3 cells, a representative in vitro model of bronchial epithelium, are used for comparison. Transporter expression has been evaluated with RT-qPCR and Western blot, the localization with immunocytochemistry, and the activity by measuring the apical-to-basolateral and basolateral-to-apical fluxes of specific substrates in the presence of inhibitors. EpiAirway™ and Calu-3 cells express high levels of MRP1 on the basolateral membrane, while they profoundly differ in terms of BCRP and MDR1: BCRP is detected in EpiAirway™, but not in Calu-3 cells, while MDR1 is expressed and functional only in fully-differentiated Calu-3; in EpiAirway™, MDR1 expression and activity are undetectable, consistently with the absence of the protein in specimens from human healthy bronchi. In summary, EpiAirway™ appears to be a promising tool to study the mechanisms of drug delivery in the bronchial epithelium and to clarify the role of ABC transporters in the modulation of the bioavailability of administered drugs.

The importance of glutamine (Gln) metabolism in multiple myeloma (MM) cells and its potential role as a therapeutic target are still unknown, although it has been reported that human myeloma cell lines (HMCLs) are highly sensitive to Gln depletion. In this study, we found that both HMCLs and primary bone marrow (BM) CD138(+) cells produced large amounts of ammonium in the presence of Gln. MM patients have lower BM plasma Gln with higher ammonium and glutamate than patients with indolent monoclonal gammopathies. Interestingly, HMCLs expressed glutaminase (GLS1) and were sensitive to its inhibition, whereas they exhibited negligible expression of glutamine synthetase (GS). High GLS1 and low GS expression were also observed in primary CD138(+) cells. Gln-free incubation or treatment with the glutaminolytic enzyme l-asparaginase depleted the cell contents of Gln, glutamate, and the anaplerotic substrate 2-oxoglutarate, inhibiting MM cell growth. Consistent with the dependence of MM cells on extracellular Gln, a gene expression profile analysis, on both proprietary and published datasets, showed an increased expression of the Gln transporters SNAT1, ASCT2, and LAT1 by CD138(+) cells across the progression of monoclonal gammopathies. Among these transporters, only ASCT2 inhibition in HMCLs caused a marked decrease in Gln uptake and a significant fall in cell growth. Consistently, stable ASCT2 downregulation by a lentiviral approach inhibited HMCL growth in vitro and in a murine model. In conclusion, MM cells strictly depend on extracellular Gln and show features of Gln addiction. Therefore, the inhibition of Gln uptake is a new attractive therapeutic strategy for MM.

Celiac disease (CD) is an autoimmune enteropathy triggered in susceptible individuals by the ingestion of gliadin-containing grains. Recent studies have demonstrated that macrophages play a key role in the pathogenesis of CD through the release of inflammatory mediators such as cytokines and nitric oxide (NO). Since arginine is the obliged substrate of iNOS (inducible nitric oxide synthase), the enzyme that produces large amount of NO, the aim of this work is to investigate arginine metabolic pathways in RAW264.7 murine macrophages after treatment with PT-gliadin (PTG) in the absence and in the presence of IFNγ. Our results demonstrate that, besides strengthening the IFNγ-dependent activation of iNOS, gliadin is also an inducer of arginase, the enzyme that transforms arginine into ornithine and urea. Gliadin treatment increases, indeed, the expression and the activity of arginase, leading to the production of polyamines through the subsequent induction of ornithine decarboxylase. This effect is strengthened by IFNγ. The activation of these pathways takes advantage of the increased availability of arginine due to a decreased system y+l-mediated efflux, likely ascribable to a reduced expression of Slc7a6 transporter. A significant induction of arginase expression is also observed in human monocytes from healthy subject upon treatment with gliadin, thus demonstrating that gluten components trigger changes in arginine metabolism in monocyte/macrophage cells.

Celiac disease (CD) is an immune-mediated enteropathy triggered by ingested gluten in genetically susceptible individuals and sustained by both adaptive and innate immune responses. Recent studies in murine macrophages demonstrated that the activation of arginase (ARG) metabolic pathway by gluten peptides contributes to the modulation of intestinal permeability in vitro. Here we characterize the effects of gluten on arginine metabolism and cell polarization in human monocytes from both healthy and CD subjects; both a simplified enzymatic digestion of gliadin and a physiological digestion of whole wheat have been tested. Results indicate that gluten digests induce the onset of an M2-like phenotype in activated macrophages; more precisely, both isoforms of arginase, ARG1 and ARG2, are induced likely due to the inhibition of mTOR and the consequent induction of C/EBPβ transcription factor. These effects are independent from the origin of gluten as well as from the digestive protocol employed; moreover, no statistical difference can be evidenced between healthy and CD patients, excluding a diverse predisposition of CD monocytes to gluten-triggered polarization with respect to healthy immune cells. Overall, the present findings sustain a role for arginase pathway in the immune response elicited by human monocytes toward ingested gluten that, hence, deserves particular attention when addressing the pathogenesis of CD.

Arginine is a semi-essential amino acid that plays an important role in the regulation of metabolic processes associated with several pathological/physiological conditions. In the vasculature, it mainly exerts its biological functions as a substrate of two alternative pathways: the conversion to nitric oxide (NO) by nitric oxide synthase (NOS) and the breakdown to urea and ornithine by arginase. To determine arginine metabolism, in the current study we propose an original radiochemical technique that allows the simultaneous monitoring of NOS and arginase activation within intact cells. Taking advantage of this method, we show here the consequences of different experimental conditions known to modulate endothelial homeostasis on arginine metabolism.

Lysinuric Protein Intolerance (LPI, MIM 222700) is a recessive aminoaciduria caused by defective cationic amino acid transport in epithelial cells of intestine and kidney. SLC7A7, the gene mutated in LPI, codifies for the y+LAT1 subunit of system y(+)L amino acid transporter. LPI patients frequently display severe complications, such as pulmonary disease, haematological abnormalities and disorders of the immune response. The transport defect may explain only a part of the clinical aspects of the disease, while the mechanisms linking the genetic defect to the clinical features of the patients remain thus far obscure. The aim of the study is to investigate the consequences of SLC7A7 mutations on specific macrophage functions, so as to evaluate if a macrophage dysfunction may have a role in the development of pulmonary and immunological complications of LPI. The results presented 1) confirm previous data obtained in one LPI patient, demonstrating that arginine influx through system y(+)L is markedly compromised in LPI macrophages; 2) demonstrate that also system y(+)L-mediated arginine efflux is significantly lower in LPI macrophages than in normal cells and 3) demonstrate that the phagocytic activity of LPI macrophages is severely impaired. In conclusion, SLC7A7/y+LAT1 mutations lead to a defective phenotype of macrophages, supporting the pathogenetic role of these cells in the development of LPI-associated complications.

Endothelial metabolism of arginine plays a key role in vascular homeostasis. While it is documented that the availability of extracellular arginine is critical for nitric oxide synthesis by eNOS, little is known about the relationships existing between arginine transport and the activity of arginase, the enzyme responsible for the production of ornithine and urea. The present study aims to characterize the role of PKC in the regulation of arginine transport and metabolism by human umbilical vein (HUVEC) and aortic (HAEC) endothelial cells. The results obtained demonstrate that the activation of PKCalpha by phorbol esters or thymeleatoxin causes a transient increase of arginine transport through system y(+), referable to the induction of SLC7A2 mRNAs and to the increased expression of CAT2 transporters. PKCalpha-dependent stimulation of arginine transport requires the activation of MEK/ERK1/2 cascade, which leads to the stimulation of AP-1 and to the consequent induction of CAT2 expression. In parallel, PKCalpha activation also increases arginase expression and activity and promotes eNOS phosphorylation, resulting in decreased NO production. It is concluded that the activation of PKCalpha stimulates arginine entry in human endothelial cells and shifts the metabolism of the cationic amino acid from NO synthesis to arginase-dependent production of ornithine and urea. This metabolic deviation may contribute to the endothelial dysfunction associated with conditions of PKC overactivity.

In endothelial cells Tumor Necrosis Factor-alpha (TNFalpha) stimulates arginine transport through the increased expression of SLC7A2/CAT2 transcripts. Here we show that also rapamycin, an inhibitor of mTOR kinase, stimulates system y(+)-mediated arginine uptake in human endothelial cells derived from either saphenous (HSVECs) or umbilical veins (HUVECs). When used together with TNFalpha, rapamycin produces an additive stimulation of arginine transport in both cell models. These effects are observed also upon incubation with AICAR, a stimulator of Adenosine-Monophosphate-dependent-Protein Kinase (AMPK) that produces a rapamycin-independent inhibition of the mTOR pathway. Rapamycin increases the V(max) of high affinity arginine transport and causes the appearance of a low affinity component that is particularly evident if the treatment is carried out in the presence of TNFalpha. RT-qPCR studies have demonstrated that these kinetic changes correspond to the induction of both the high affinity transporter CAT2B and the low affinity isoform CAT2A. Western blot and immunocytochemical analyses indicate that, consistently, the expression of CAT2 proteins is also stimulated under the same conditions. These changes are associated with an increase of the intracellular arginine concentration but with a decrease of NO production. Thus, our data suggest that mTOR activity is associated with the repression of CAT2 expression at mRNA and protein level.

In this study we have employed three lines of C127 murine cells. C127 CFTR w/t, C127 CFTR delta F508 and C127 mock, transfected with, respectively, wild type, delta F508 mutant human CFTR cDNA or the vector only. In the first 10 minutes of a Cl(-)-free incubation the three cell lines exhibit a significant shrinkage due to a loss of K+ and Cl-. However, C127 CFTR w/t cells shrink more than C127 CFTR delta F508 and the mock cells. The supplementation of Cl(-)-free medium with ATP causes a marked decrease in the cell volume of C127 CFTR delta F508 and of the mock cells but not of C127 CFTR w/t cells. ATP effect is mimicked by adenosine 5'-O-(3-thiotriphosphate), but neither by adenosine nor by UTP. Measurements of extracellular ATP indicate that during the Cl(-)-free incubation C127 CFTR w/t cells extrude more ATP than the other two cell lines. The results are consistent with the hypothesis that CFTR enhances K+ and Cl- permeabilities by promoting the extrusion of ATP.

Regulatory volume decrease (RVD) has been studied in cultured human fibroblasts incubated in a complete growth medium at low osmolality (215 mosmolal). After the initial swelling induced by hypotonic treatment, cells recover their volume almost completely within about 60 min. This RVD is associated with comparable losses of cell potassium and amino acids. After an initial increase, cell content of sodium is kept at values close to control. Chromatographic analysis of intracellular amino-acid pool has shown that RVD-associated decrease in cell amino acids is due for the most part to changes in the intracellular concentration of L-glutamine. RVD-exerting cells undergo a rapid and marked depolarization that is maintained after cell volume recovery. This change in membrane potential has been detected with measurements of both the transmembrane distribution ratios of L-arginine and of fluorescence of potential-sensitive dye bis-oxonol. Due to depolarization, the trans-membrane gradient of sodium electrochemical potential is lowered. It is proposed that cell depolarization concurs to keep the intracellular concentration of amino acids low by inhibiting sodium-coupled uptake through system A.

Background In the recessive aminoaciduria Lysinuric Protein Intolerance (LPI), mutations of SLC7A7/y+LAT1 impair system y+L transport activity for cationic amino acids. A severe complication of LPI is a form of Pulmonary Alveolar Proteinosis (PAP), in which alveolar spaces are filled with lipoproteinaceous material because of the impaired surfactant clearance by resident macrophages. The pathogenesis of LPI-associated PAP remains still obscure. The present study investigates for the first time the expression and function of y+LAT1 in monocytes and macrophages isolated from a patient affected by LPI-associated PAP. A comparison with mesenchymal cells from the same subject has been also performed. Methods Monocytes from peripheral blood were isolated from a 21-year-old patient with LPI. Alveolar macrophages and fibroblastic-like mesenchymal cells were obtained from a whole lung lavage (WLL) performed on the same patient. System y+L activity was determined measuring the 1-min uptake of [3H]-arginine under discriminating conditions. Gene expression was evaluated through qRT-PCR. Results We have found that: 1) system y+L activity is markedly lowered in monocytes and alveolar macrophages from the LPI patient, because of the prevailing expression of SLC7A7/y+LAT1 in these cells; 2) on the contrary, fibroblasts isolated from the same patient do not display the transport defect due to compensation by the SLC7A6/y+LAT2 isoform; 3) in both normal and LPI monocytes, GM-CSF induces the expression of SLC7A7, suggesting that the gene is a target of the cytokine; 4) GM-CSF-induced differentiation of LPI monocytes is comparable to that of normal cells, demonstrating that GM-CSF signalling is unaltered; 5) general and respiratory conditions of the patient, along with PAP-associated parameters, markedly improved after GM-CSF therapy through aerosolization. Conclusions Monocytes and macrophages, but not fibroblasts, derived from a LPI patient clearly display the defect in system y+L-mediated arginine transport. The different transport phenotypes are referable to the relative levels of expression of SLC7A7 and SLC7A6. Moreover, the expression of SLC7A7 is regulated by GM-CSF in monocytes, pointing to a role of y+LAT1 in the pathogenesis of LPI associated PAP.

The influx of L-threonine through system ASC does not influence the membrane potential in cultured human fibroblasts although comparable fluxes of amino acids through another Na(+)-dependent agency, system A, effectively depolarize the cells. The membrane potential, however, stimulates the influx of amino acids through system ASC with a maximal effect at -50 mV. The sensitivity of amino acid influx through system ASC to the membrane potential is not constant, but rather, is dependent on intracellular and extracellular concentrations of the substrates, Na+ and amino acids, of the system. Conditions which favor the loading of the ASC carrier at the external surface reduce the sensitivity of ASC-mediated amino acid influx to the membrane potential; in contrast, the sensitivity of this amino acid influx increases under conditions which favor loading of the carrier at the internal surface. Trans-stimulation, a well-known characteristic of system ASC, also varies with the concentrations of the substrates of the system and, in fact, this characteristic is not observed when external Na+ is low. These data may be accommodated by a model in which an electrically silent mode of operation of the transporter is dominant. The influence of the membrane potential on the transport system is dependent on the extent to which a charge-translocating step in the cycling of the carrier is rate limiting (relative rate limitance).

The energization of System A in cultured human fibroblasts has been studied by measuring the energy transfer from the electrochemical gradient of Na+ to the chemical gradient of the site A-specific substrate amino acid 2-methylaminoisobutyric acid. The co-transport Na+/amino acid, studied by kinetic analysis and radiochemical measurements, showed a coupling ratio of 1:1. The assessment of the Na+ electrochemical gradient in cultured adherent cells relied on the development of noninvasive procedures as follows: the membrane electrical potential was estimated from the accumulation of L-arginine at equilibrium (Bussolati, O., Laris, P. C., Nucci, F. A., Dall'Asta, V., Longo, N., Guidotti, G. G., and Gazzola, G. C. (1987) Am. J. Physiol. 253, C391-C397); the chemical gradient of Na+ was determined from spectrometric measurements of Na+. The accumulation of 2-methylaminoisobutyric acid was strongly sensitive to changes of Na+ gradient and of membrane electrical potential, indicating that the electrochemical gradient of Na+ contributed energy for the uphill transport of the amino acid through System A. Changes in the Na+ electrochemical gradient were obtained by: (i) alterations of extracellular concentration of Na+; (ii) changes of membrane electrical potential obtained by variation of extracellular [K+]; and (iii) changes of [Na+]in and membrane electrical potential upon incubation of the cells in serum-free saline solutions (Dall'Asta, V., Gazzola, G. C., Longo, N., Bussolati, O., Franchi-Gazzola, R., and Guidotti, G. G. (1986) Biochim. Biophys. Acta 860, 1-8). The correlation between the chemical gradient of 2-methylaminoisobutyric acid and the Na+ electrochemical potential followed a straight line with a yield close to the thermodynamic equilibrium, thus suggesting that the energy stored in the gradient of Na+ electrochemical potential is fully adequate to energize the intracellular accumulation of site A-reactive amino acids in human fibroblasts.

Aim Drug-eluting stents are widely used to prevent restenosis but are associated with late endothelial damage. To understand the basis for this effect, we have studied the consequences of a prolonged incubation with rapamycin on the viability and functions of endothelial cells. Methods and results Human umbilical vein or aorta endothelial cells were exposed to rapamycin in the absence or in the presence of tumour necrosis factor α (TNFα). After a 24 h-incubation, rapamycin (100 nM) caused a significant cell loss associated with the increase of both apoptosis and necrosis, as quantified by propidium iodide staining, caspase 3 activity, and lactate dehydrogenase release. Rapamycin also impaired cell mobility, as assessed by a wound test, and promoted the formation of actin stress fibres, as determined with confocal microscopy. Moreover, the inhibitor prolonged TNFα-dependent E-selectin induction, inhibited endothelial nitric oxide synthase expression at both mRNA (quantitative real-time polymerase chain reaction) and protein level (enzyme-linked immunosorbent assay and western blot), and lowered bioactive nitric oxide output (RFL-6 reporter cell assay). Under the conditions adopted, rapamycin inhibited both mammalian target-of-rapamycin complexes (mTORC1 and mTORC2), as indicated by the reduced amount of raptor and rictor bound to mTOR in immunoprecipitates and by the marked hypophosphorylation of protein S6 kinase I (p70S6K) and Akt, determined by western blotting. The selective inhibition of mTORC1 by AICAR did not affect endothelial viability. Conclusion A prolonged treatment with rapamycin impairs endothelial function and hinders cell viability. Endothelial damage seems dependent on mTORC2 inhibition.

The transport of l -glutamine has been studied in diploid human fibroblasts in culture. Mathematical discrimination by nonlinear regression, competition analysis, and conditions varying the relative contribution of the various mediations have been used to characterize the systems engaged in the inward transport of this amino acid. The adopted criteria showed that l -glutamine enters the fibroblast by the Na + -dependent systems ASC and A and by a Na + -independent route identified as system L. The relative contribution of these agencies to the total saturable uptake of glutamine varied with the concentration of the amino acid and with the nutritional state of the cell. At amino acid concentrations approaching those encountered in human plasma: (1) system ASC represented the primary mediation for entry of l -glutamine in human fibroblasts; (2) the contribution of system A was lower, though significant, in unstarved repressed cells and became predominant in starved derepressed cells; (3) the Na + -independent system L accounted for less than one-fifth of glutamine uptake in either nutritional condition. The changes in the relative contribution of the various systems to the uptake of glutamine as a function of its concentration may have implications in pathophysiology under conditions associated with enhanced glutamine concentrations in the extracellular fluids.

Systems y+ and y+L represent the main routes for arginine transport in mammalian cells. While system y+ activity is needed for the stimulated NO production in rodent alveolar macrophages (AM), no information is yet available about arginine transport in human AM. We study here arginine influx and genes for arginine transporters in AM from bronchoalveolar lavage of normal subjects. These cells express the y+ -related genes SLC7A1/CAT1 and SLC7A2/CAT2B, as well as the y+L genes SLC7A7/y+LAT1 and SLC7A6/y+LAT2. However, compared with human endothelial cells, AM express much less SLC7A2 mRNA and higher levels of SLC7A7 mRNA. Granulocyte macrophage colony-stimulating factor or IFN-gamma do not change the expression of any transporter gene, while lipopolysaccharide induces SLC7A2/CAT2B. Under all the conditions tested, leucine inhibits most of the arginine transport in the presence of Na+ and N-ethylmaleimide, an inhibitor of system y+, is completely ineffective, indicating that system y+L operates most of the arginine influx. Comparable results are obtained in AM from patients with interstitial lung disease, such as Nonspecific Interstitial Pneumonia (NSIP), although these cells have a higher SLC7A1 and a lower SLC7A7 expression than AM from normal subjects. It is concluded that AM from normal subjects or patients with NSIP lack a functional transport system y+, a situation that may limit arginine availability for NO synthesis. Moreover, since mutations of SLC7A7/y+LAT1 cause Lysinuric Protein Intolerance, a disease often associated with AM impairment and alveolar proteinosis, the high SLC7A7 expression observed in human AM suggests that y+LAT1 activity is important for the function of these cells.

Under hypertonic conditions the induction of SLC38A2/SNAT2 leads to the stimulation of transport system A and to the increase in the cell content of amino acids. In hypertonically stressed human fibroblasts transfection with two siRNAs for SNAT2 suppressed the increase in SNAT2 mRNA and the stimulation of system A transport activity. Under the same condition, the expansion of the intracellular amino acid pool was significantly lowered and cell volume recovery markedly delayed. It is concluded that the up-regulation of SNAT2 is essential for the rapid restoration of cell volume after hypertonic stress.

The resistance to L-asparaginase (ASNase) has been associated to the overexpression of asparagine synthetase (AS), although the role played by other metabolic adaptations has not been yet defined. Both in ASNase-sensitive Jensen rat sarcoma cells and in ARJ cells, their ASNase-resistant counterparts endowed with a five-fold increased AS activity, ASNase treatment rapidly depletes intracellular asparagine. Under these conditions, cell glutamine is also severely reduced and the activity of glutamine synthetase (GS) is very low. After 24 h of treatment, while sensitive cells have undergone massive apoptosis, ARJ cells exhibit a marked increase in GS activity, associated with overexpression of GS protein but not of GS mRNA, and a partial restoration of glutamine and asparagine. However, when ARJ cells are treated with both ASNase and L-methionine-sulfoximine (MSO), an inhibitor of GS, no restoration of cell amino acids occurs and the cell population undergoes a typical apoptosis. No toxicity is observed upon MSO treatment in the absence of ASNase. The effects of MSO are not referable to depletion of cell glutathione or inhibition of AS. These findings indicate that, in the presence of ASNase, the inhibition of GS triggers apoptosis. GS may thus constitute a target for the suppression of ASNase-resistant phenotypes.

System A is a secondary active, sodium dependent transport system for neutral amino acids. Strictly coupled with Na,K-ATPase, its activity determines the size of the intracellular amino acid pool, through a complex network of metabolic reaction and exchange fluxes. Many hormones and drugs affect system A activity in specific cell models or tissues. In all the cell models tested thus far the activity of the system is stimulated by amino acid starvation, cell cycle progression, and the incubation under hypertonic conditions. These three conditions produce marked alterations of cell volume. The stimulation of system A activity plays an important role in cell volume restoration, through an expansion of the intracellular amino acid pool. Under normal conditions, system A substrates represent a major fraction of cell compatible osmolytes, organic compounds that exert a protein stabilizing effect. It is, therefore, likely that the activation of system A represents a portion of a more complex response triggered by exposure to stresses of various nature. Since system A transporters have been recently cloned, the molecular bases of these regulatory mechanisms will probably be elucidated in a short time.

Amino acid starvation markedly stimulates the activity of system A, a widely distributed transport route for neutral amino acids. The involvement of MAPK (mitogen-activated protein kinase) pathways in this adaptive increase of transport activity was studied in cultured human fibroblasts. In these cells, a 3-fold stimulation of system A transport activity required a 6-h amino acid-free incubation. However, a rapid tyrosine phosphorylation of ERK (extracellular regulated kinase) 1 and 2, and JNK (Jun N-terminal kinase) 1, but not of p38, was observed after the substitution of complete medium with amino acid-free saline solution. ERK1/2 activity was 4-fold enhanced after a 15-min amino acid-free incubation and maintained at stimulated values thereafter. A transient, less evident stimulation of JNK1 activity was also detected, while the activity of p38 was not affected by amino acid deprivation. PD98059, an inhibitor of ERK1/2 activation, completely suppressed the adaptive increase of system A transport activity that, conversely, was unaffected by inhibitors of other transduction pathways, such as rapamycin and wortmannin, as well as by chronic treatment with phorbol esters. In the presence of either L-proline or 2-(methylaminoisobutyric) acid, two substrates of system A, the transport increase was prevented and no sustained stimulation of ERK1/2 was observed. To identify the stimulus that maintains MAPK activation, cell volume was monitored during amino acid-free incubation. It was found that amino acid deprivation caused a progressive cell shrinkage (30% after a 6-h starvation). If proline was added to amino acid-starved, shrunken cells, normal values of cell volume were rapidly restored. However, proline-dependent volume rescue was hampered if cells were pretreated with PD98059. It is concluded that (a) the triggering of adaptive increase of system A activity requires a prolonged activation of ERK1 and 2 and that (b) cell volume changes, caused by the depletion of intracellular amino acid pool, may underlie the activation of MAPKs.

It has been known for several years that the triggering of cell proliferation is associated with an increase of the activity of Na,K,Cl cotransport and of transport system A for neutral amino acids. These systems are also enhanced during the volume recovery of hypertonically shrunk cells. We demonstrate here that during the cell cycle of NIH3T3 cells, an increase in cell volume is associated with an enhanced cell content of potassium and amino acids. Bumetanide delays cell cycle progression and hampers volume increase. The nonmetabolizable analog 2-methylamino-isobutyric acid, a specific substrate of system A, can partially substitute natural amino acids accumulated during the cell cycle as intracellular osmolytes. It is therefore proposed that the stimulation of Na,K,Cl cotransport and of system A, observed in proliferating cells, causes an expansion of cell volume through an enhanced intracellular accumulation of both inorganic and organic osmolytes and the concurrent, osmotically obliged uptake of water.

When the expression of a Ha-ras oncogene is triggered in NIH3T3 cells, a progressive inhibition of sodium dependent transport of anionic amino acids through system X-AG is observed. After 48 h of ras expression the transport activity of system X-AG is almost abolished, while other transport systems involved in anionic amino acid transport are unaffected or even stimulated. In the presence of high extracellular concentrations of glutamine, the intracellular concentration of glutamate is comparable in ras expressing and non-expressing cells. On the contrary, when the extracellular pool of glutamine is depleted by the enzyme L-asparaginase, intracellular glutamate decreases at a much faster rate in ras expressing, low-transport cells. These results suggest that transport system X-AG significantly contributes to the homeostasis of intracellular glutamate under conditions of glutamine deprivation.

Using L-cysteate and tcysteinesulfinate as model substrates, we characterize here a transport system, both in cultured rat hepatocytes and human skin fibroblasts, serving for the anions glutamate and aspartate, but not for the dipolar species glutamic and aspartic acids. This system appears to be accompanied by a second, lower affinity system for the anionic forms, which is also Na’-dependent; this lower affinity system applies at least to glutamate. These systems show the usual degree of preference for L- over D-glutamate and, in the fibroblast, for L- over DL-a-aminoadipate. D-ASpartate proved nearly as inhibitory to the uptake of Lcysteate or t-aspartate, however, as did L-aspartate itself, a comparison recalling a similar stereoselective anomaly discovered by Pall in Neurospora (Pall, M. (1976) Biochim Biophys. Acta 211, 513-520). We conclude that this anomaly arises from the ability of the two substrate carboxylate groups to bond in the spatial order either a# for the L-isomer or &a for the D-isomer and also to bond in the order a,y for L-glutamate, but scarcely in the order y,a for D-glutamate. A major lack of inhibition by D-cysteate, which might be expected to bind like aspartate in the inverted order, shows, however, that the two anionic groups are not recognized in identical manners by the two corresponding subsites, Precedent for a chemical difference in these two subsites is available from transport systems for neutral aand p-amino acids. A strong transport inhibition of the hepatocyte system by 3-aminoglutarate shows that an a,a relation between the amino group and either of the carboxylate groups of the anionic amino acid is not required. The above anomaly in stereoselectivity is compared with a corresponding one, applying to the reactions of aspartic acid and asparagine, versus glutamic acid and glutamine, with System L for neutral amino acid transport in the Ehrlich cell. A weak pHdependent inhibition of the uptake of anionic amino acids by cysteine can be associated with its unique mode of conversion to an anionic species.

The transport of L-arginine has been characterized in Chinese hamster ovary cells (CHO). In the absence of Na+ the influx of the amino acid decreased. Both in the presence and in the absence of Na+ L-arginine influx was trans-stimulated and cis-inhibited by cationic amino acids. The amino acid entered CHO cells through an apparently non saturable mechanism and a single saturable agency whose Km increased in the absence of Na+. These results indicate that the agency devoted to transport cationic amino acids in CHO cells resembles system y+, the Na+-independent route that transports cationic amino acids in a number of mammalian models, although its activity is lowered by the replacement of extracellular sodium.

The transport of neutral amino acids and their interactions for uptake have been studied in skin-derived-diploid human fibroblasts cultured at confluency. Properly timed preincubations in a medium defined in composition and requirements have been adopted to control size of internal amino acid pools and regulatory interferences affecting amino acid transport. L-Proline, L-alanine, and L-leucine were used as natural amino acid substrates. Amino acid uptake and exchange with preaccumulated molecules have been measured under conditions approaching initial entry rates in the absence and in the presence of Na+ and of transport-specific model substrates. Cultured human fibroblasts were found to contain three neutral amino acid transport systems: A, ASC, and L. L-Proline was taken up preferentially by System A. System ASC appeared to mediate the largest fraction of L-Leucine entered the cells mainly by the Na+-independent System L. Preaccumulation of the cells with Site A-reactive amino acids caused trans-inhibition of the activity of System A. The activity of System L was strongly trans-stimulated in cells preloaded with Site L-reactive amino acids. The inward transport of L-alanine was trans-inhibited by internal Site A-reactive amino acids and trans-stimulated by preaccumulated L-alanine exchanging with the external amino acid through the operation of System ASC.

The regulation of the transport of neutral amino acids across the cell membrane by adaptive mechanisms has been studied in cultured human fibroblasts. Among the three transport systems (A, ASC, and L) individually discriminated, only the Na+-dependent System A was subject to adaptive regulation, showing enhancement of its activity when the cells were incubated under conditions of amino acid shortage (derepression phase) and decrease of its activity when the cells were exposed to a medium supplied with Site A-reactive amino acids (repression phase). Starvation-induced derepression of transport activity and its reversal by amino acid refeeding required active RNA and protein synthesis. Derepression involved an early mRNA synthesis which started within 30 min from the abrupt change in extracellular amino acid concentration and apparently lasted 90 min. The transcribed mRNA was rather stable and translatable for a few hours (presumably into transport proteins) in cells maintained in the absence of amino acids. Repression by amino acid refeeding also involved an early mRNA synthesis, the product of its translation being presumably a protein capable of causing degradation or inactivation of transport proteins. The rate of decay in transport activity of previously derepressed cells was somewhat faster in the presence of added Site A-reactive amino acids than in their absence. A model is proposed in which the concentration of Site A-reactive amino acids affects transport activity of System A by modulating transcription of mRNA species coding for transport proteins and their putative inactivators and by regulating the efficiency of transport protein inactivation at the cell membrane.

Summary The transport of glycine was studied in cultured human fibroblasts. The amino acid entered the cell by Na + -dependent and Na + -independent mechanisms. Na + -independent glycine (0.1 mM) transport was less than 10% of total uptake and occurred by a mechanism formally indistinguishable from diffusion. Two distinct routes contributed to Na + -dependent glycine transport. The first route was identified with system A because it was inhibited by MeAIB and underwent adaptive regulation. The second route was identified with system ASC as it was inhibited by L-alanine, but not by MeAIB. Kinetic analysis revealed that the two systems operated glycine transport with the same Km of 1.6 mM, a value unusually high for system ASC.

The action of serum on the expression of the starvation-enhanced amino acid transport by System A (as a part of the adaptive regulation mechanism) has been studied in cultured fetal human fibroblasts. Serum enhanced L-proline uptake of cells starved in serum-free medium. This effect was rapid, proportional to the amount of pre-existing transporters, insensitive to cycloheximide and kinetically characterized by an increase of transport Vmax. These results can be interpreted to indicate that serum is essential for a vectorial post-translational event leading to insertion of transport proteins into the cell membrane.

The transport of L-alanine, L-serine, and L-cysteine has been studied in skin-derived diploid human fibroblasts in culture. Competition analysis, mathematical discrimination by nonlinear regression, and conditions varying the relative contribution of the various mediations have been used to characterize the systems engaged in the inward transport of these amino acids. All the adopted criteria yielded results showing that L-alanine, L-serine, and L-cysteine enter the cell by two Na+-dependent systems, System A and System ASC, and by a Na+-independent route, whose major component has been identified as System L. The apparent affinity of L-alanine, L-serine, and L-cysteine for the putative carrier was higher for System ASC than for System A. The transport Vmax for System A increased in response to cell starvation; after 12 h, its values were similar or higher than those exhibited by System ASC. At amino acid concentrations approaching those present in human plasma, System ASC appeared to be the primary mediation for the inward transport of L-alanine, L-serine, and L-cysteine in human fibroblasts. The contribution of System A was negligible in nonstarved cells and became appreciable under conditions of cell starvation. The Na+-independent System L made no substantial contribution to the uptake of L-alanine and L-serine and accounted for approximately one-fourth of the total uptake of L-cysteine.

The use of an inexpensive and simple modification of Costar 24-well cluster trays is described in a rapid and reproducible method for measuring substrate fluxes in adherent cultured eukaryotic cells.

Freshly isolated human monocytes transport L-arginine mostly through a sodium independent, NEM insensitive pathway inhibited by L-leucine in the presence, but not in the absence of sodium. Interferon-gamma (IFNgamma) stimulates this pathway, identifiable with system y+L, and markedly enhances the expression of SLC7A7, the gene that encodes for system y+L subunit y+LAT1, but not of SLC7A6, that codes for the alternative subunit y+LAT2. System y+ plays a minor role in arginine uptake by monocytes and the expression of system y+-related genes, SLC7A1 and SLC7A2, is not changed by IFNgamma. These results demonstrate that system y+L is sensitive to IFNgamma.

Celiac disease (CD) is an immune-mediated enteropathy sustained by dietary gluten in susceptible individuals, and characterized by a complex interplay between adaptive and innate responses against gluten peptides (PTG). In a recent contribution we have demonstrated that the treatment with PTG induces the expression and activity of arginase in both murine macrophages and human monocytes from healthy subjects, thus suggesting a role for arginine and its metabolites in gluten-triggered response of these cells. Here we further explore this field, by addressing the effects of PTG on polyamine synthesis and release in murine RAW264.7 macrophages, and how they affect epithelial permeability of Caco-2 monolayers. Results obtained show a massive production and release of putrescine by macrophages upon incubation with gluten peptides; this, in turn, causes a decrease in TEER in epithelial cells, indicating that PTG-driven secretion of polyamines by macrophages has a role in the modulation of intestinal permeability in vitro. At a molecular level, putrescine production appears referable to the activation of C/EBPβ transcription factor, which is known to be responsible for arginase induction in activated macrophages and is a crucial mediator of inflammation. Whether these pathways are stimulated also in vivo deserves to be further investigated, as well as their role in gluten-driven cellular and intestinal defects typical of CD patients.

The regulation of amino acid transport by insulin has been studied in cultured human fibroblasts. Among the six amino acid transport systems operating in cultured human fibroblasts, two systems (A and x−C) are strongly stimulated by insulin and four (ASC, X−AG, y+ and L) are essentially not sensitive to the presence of the hormone in the incubation medium. The hormonal stimulation of system A and system x−C became significant after 3 h of incubation and increased up to 12 h. The stimulatory effect was related to insulin concentration, with a half-maximal stimulation at 10−9 M hormone concentration. Insulin enhanced transport activity by increasing the maximal velocity (Vmax) of transport, without significant changes in Km values.

The transport of L-glutamic acid has been studied in skin-derived diploid human fibroblasts. Competition analysis in the presence and absence of Na+ and mathematical discrimination by nonlinear regression indicated that L-glutamic acid enters the cell by at least three transport systems: 1) a high affinity Na+-dependent system which has been found to be identical to the previously described system for anionic amino acids (Gazzola, G. C., Dall'Asta, V., Bussolati, O., Makowske, M., and Christensen, H. N. (1981) J. Biol. Chem. 256, 6054-6059) and which is provisionally designated as System X-AG; this route was shared by L-aspartic acid; 2) a low affinity Na+-dependent system resembling the ASC System for neutral amino acids (Franchi-Gazzola, R., Gazzola, G. C., Dall'Asta, V., and Guidotti, G. G. (1982) J. Biol. Chem. 257, 9582-9587); its reactivity toward L-glutamic acid was strongly inhibited by L-serine, but not by 2-(methyl-amino)isobutyric acid; and 3) a Na+-independent system similar to System XC- described in fetal human lung fibroblasts (Bannai, S., and Kitamura, E. (1980) J. Biol. Chem. 255, 2372-2376). The XC- system served for L-glutamic acid and L-cystine, the latter amino acid behaving as a potent inhibitor of L-glutamic acid uptake. Amino acid starvation did not change the uptake of L-glutamic acid by the two Na+-dependent systems, but enhanced the activity of System XC- by increasing its Vmax. L-Glutamic acid transport was also affected by the density of the culture. An increased cell density lowered the uptake of the amino acid by Systems ASC and XC- and promoted the uptake by System X-AG. All these variations were dependent upon changes in Vmax.

Human umbilical vein endothelial cells transport arginine through two Na(+)-independent systems. System y(+)L is insensitive to N-ethylmaleimide (NEM), inhibited by L-leucine in the presence of Na(+), and referable to the expression of SLC7A6/y(+)LAT2, SLC7A7/y(+)LAT1, and SLC3A2/4F2hc. System y(+) is referable to the expression of SLC7A1/CAT1 and SLC7A2/CAT2B. Tumor necrosis factor-alpha (TNF-alpha) and bacterial lipopolysaccharide induce a transient stimulation of arginine influx and efflux through system y(+). Increased expression of SLC7A2/CAT2B is detectable from 3 h of treatment, while SLC7A1 expression is inhibited at later times of incubation. System y(+)L activity and expression remain unaltered. Nitric oxide synthase type 2 mRNA is not detected in the absence or presence of TNF-alpha, while the latter condition lowers nitric oxide synthase type 3 expression at the mRNA and the protein level. Nitrite accumulation is comparable in cytokine-treated and control cells up to 48 h of treatment. It is concluded that modulation of endothelial arginine transport by TNF-alpha or lipopolysaccharide occurs exclusively through changes in CAT2B and CAT1 expression and is dissociated from stimulation of nitric oxide production. Human umbilical vein endothelial cells transport arginine through two Na+-independent systems. System y+L is insensitive to N-ethylmaleimide (NEM), inhibited by L-leucine in the presence of Na+, and referable to the expression of SLC7A6/y+LAT2, SLC7A7/y+LAT1, and SLC3A2/4F2hc. System y+ is referable to the expression of SLC7A1/CAT1 and SLC7A2/CAT2B. Tumor necrosis factor-α (TNF-α) and bacterial lipopolysaccharide induce a transient stimulation of arginine influx and efflux through system y+. Increased expression of SLC7A2/CAT2B is detectable from 3 h of treatment, while SLC7A1 expression is inhibited at later times of incubation. System y+L activity and expression remain unaltered. Nitric oxide synthase type 2 mRNA is not detected in the absence or presence of TNF-α, while the latter condition lowers nitric oxide synthase type 3 expression at the mRNA and the protein level. Nitrite accumulation is comparable in cytokine-treated and control cells up to 48 h of treatment. It is concluded that modulation of endothelial arginine transport by TNF-α or lipopolysaccharide occurs exclusively through changes in CAT2B and CAT1 expression and is dissociated from stimulation of nitric oxide production.