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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.

Background. Emerging evidences suggest that in severe COVID-19, multi-organ failure is associated with a hyperinflammatory state (the so-called “cytokine storm”) in combination with the development of a prothrombotic state. The central role of endothelial dysfunction in the pathogenesis of the disease is to date accepted, but the precise mechanisms underlying the associated coagulopathy remain unclear. Whether the alterations in vascular homeostasis directly depend upon the SARS-CoV-2 infection of endothelial cells or, rather, occur secondarily to the activation of the inflammatory response is still a matter of debate. Here, we address the effect of the SARS-CoV-2 spike S1 protein on the activation of human lung microvascular endothelial cells (HLMVEC). In particular, the existence of an endothelium-macrophage crosstalk in the response to the spike protein has been explored. Methods and Results. The effect of the spike protein is addressed in human lung microvascular endothelial cells (HLMVEC), either directly or after incubation with a conditioned medium (CM) of human monocyte-derived macrophages (MDM) previously activated by the spike S1 protein (CM-MDM). Both MDM and HLMVEC are activated in response to the S1 protein, with an increased expression of pro-inflammatory mediators. However, when HLMVEC are exposed to CM-MDM, an enhanced cell activation occurs in terms of the expression of adhesion molecules, pro-coagulant markers, and chemokines. Under this experimental condition, ICAM-1 and VCAM-1, the chemokines CXCL8/IL-8, CCL2/MCP1, and CXCL10/IP-10 as well as the protein tissue factor (TF) are markedly induced. Instead, a decrease of thrombomodulin (THBD) is observed. Conclusion. Our data suggest that pro-inflammatory mediators released by spike-activated macrophages amplify the activation of endothelial cells, likely contributing to the impairment of vascular integrity and to the development of a pro-coagulative endothelium.

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.

Organic cation transporters (OCTs) and novel organic cation transporters (OCTNs) are responsible for drug delivery in the intestine and kidney, in the lung, OCTs mediate inhaled drugs&rsquo, transport, although their physiological role in airways remains poorly understood. The studies addressing OCTs/OCTNs in human airways were mostly performed in immortal or transformed cell lines, here, we studied OCTs in EpiAirway&trade, a recently developed in vitro model of normal bronchial epithelium. Calu-3 monolayers were used for comparison. The activity of OCTs was evaluated by measuring the uptake of 1-methyl-4-phenylpyridinium (MPP+) at the apical and basolateral side of monolayers and protein expression through Western Blot analysis. OCTs and OCTNs expression, along with that of Amino acid Transporter B0,+ (ATB0,+)transporter, was determined by measuring the number of mRNA molecules through quantitative Polymerase Chain Reaction (qPCR). The interaction of the transporters with bronchodilators was also assessed. Results highlight significant differences between Calu-3 cells and EpiAirway&trade, since, in the latter, OCTs are active only on the basolateral membrane where they interact with the bronchodilator ipratropium. No activity of OCTs is detectable at the apical side, there, the most abundant carrier is, instead, SLC6A14/ATB0,+, that can thus be potentially listed among organic cation transporters responsible for drug delivery in the lung.

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.

In human, OCTN2 (SLC22A5) and ATB0,+ (SLC6A14) transporters mediate the uptake of L-carnitine, essential for the transport of fatty acids into mitochondria and the subsequent degradation by β-oxidation. Aim of the present study was to characterize L-carnitine transport in EpiAirway™, a 3D organotypic in vitro model of primary human tracheal-bronchial epithelial cells that form a fully differentiated, pseudostratified columnar epithelium at air-liquid interface (ALI) condition. In parallel, Calu-3 monolayers grown at ALI for different times (8d or 21d of culture) were used as comparison. OCTN2 transporter was equally expressed in both models and functional at the basolateral side. ATB0,+ was, instead, highly expressed and active on the apical membrane of EpiAirway™ and only in early-cultures of Calu-3 (8d but not 21d ALI). In both cell models, L-carnitine uptake on the apical side was significantly inhibited by the bronchodilators glycopyrrolate and tiotropium, that hence can be considered substrates of ATB0,+; ipratropium was instead effective on the basolateral side, indicating its interaction with OCTN2. Inflammatory stimuli, such as LPS or TNFα, caused an induction of SLC6A14/ATB0,+ expression in Calu-3 cells, along with a 2-fold increase of L-carnitine uptake only at the apical side; on the contrary SLC22A5/OCTN2 was not affected. As both OCTN2 and ATB0,+, beyond transporting L-carnitine, have a significant potential as delivery systems for drugs, the identification of these transporters in EpiAirway™ can open new fields of investigation in the study of drug inhalation and pulmonary delivery.

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.

Lysinuric protein intolerance (LPI) is a recessively inherited aminoaciduria caused by mutations of SLC7A7, the gene encoding y+LAT1 light chain of system y+L for cationic amino acid transport. The pathogenesis of LPI is still unknown. In this study, we have utilized a gene silencing approach in macrophages and airway epithelial cells to investigate whether complications affecting lung and immune system are directly ascribable to the lack of SLC7A7 or, rather, mediated by an abnormal accumulation of arginine in mutated cells. When SLC7A7/y+LAT1 was silenced in human THP-1 macrophages and A549 airway epithelial cells by means of short interference RNA (siRNA), a significant induction of the expression and release of the inflammatory mediators IL1β and TNFα was observed, no matter the intracellular arginine availability. This effect was mainly regulated at transcriptional level through the activation of NFκB signaling pathway. Moreover, since respiratory epithelial cells are the important sources of chemokines in response to pro-inflammatory stimuli, the effect of IL1β has been addressed on SLC7A7 silenced A549 cells. Results obtained indicated that the downregulation of SLC7A7/y+LAT1 markedly strengthened the stimulatory effect of the cytokine on CCL5/RANTES expression and release without affecting the levels of CXCL8/IL8. Consistently, also the conditioned medium of silenced THP-1 macrophages activated airway epithelial cells in terms of CCL5/RANTES expression due to the presence of elevated amount of proinflammatory cytokines. In conclusion, our results point to a novel thus far unknown function of SLC7A7/y+LAT1, that, under physiological conditions, besides transporting arginine, may act as a brake to restrain inflammation.

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.

Background:\ud A subset of human hepatocellular carcinomas (HCC) exhibit mutations of β-catenin gene CTNNB1 and overexpress Glutamine synthetase (GS). The CTNNB1-mutated HCC cell line HepG2 is sensitive to glutamine starvation induced in vitro with the antileukemic drug Crisantaspase and the GS inhibitor methionine-L-sulfoximine (MSO).\ud \ud Methods:\ud Immunodeficient mice with subcutaneous xenografts of the CTNNB1-mutated HCC cell lines HepG2 and HC-AFW1 were treated with Crisantaspase and/or MSO, and tumour growth was monitored. At the end of treatment, tumour weight and histology were assessed. Serum and tissue amino acids were determined by HPLC. Gene and protein expression were estimated with RT-PCR and western blot and GS activity with a colorimetric method. mTOR activity was evaluated from the phosphorylation of p70S6K1.\ud \ud Results:\ud Crisantaspase and MSO depleted serum glutamine, lowered glutamine in liver and tumour tissue, and inhibited liver GS activity. HepG2 tumour growth was significantly reduced by either Crisantaspase or MSO, and completely suppressed by the combined treatment. The combined treatment was also effective against xenografts of the HC-AFW1 cell line, which is Crisantaspase resistant in vitro.\ud \ud Conclusions:\ud The combination of Crisantaspase and MSO reduces glutamine supply to CTNNB1-mutated HCC xenografts and hinders their growth.

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.

l-Carnitine, in addition to playing a fundamental role in the β-oxidation of fatty acids, has been recently identified as a modulator of immune function, although the mechanisms that underlie this role remain to be clarified. In this study, we addressed the modulation of l-carnitine transport and expression of related transporters during differentiation of human monocytes to macrophages. Whereas monocytes display a modest uptake of l-carnitine, GM-CSF–induced differentiation massively increased the saturable Na+-dependent uptake of l-carnitine. Kinetic and inhibition analyses demonstrate that in macrophage l-carnitine transport is mediated by a high-affinity component (Km ∼4 µM) that is identifiable with the operation of OCTN2 transporter and a low-affinity component (Km > 10 mM) that is identifiable with system A for neutral amino acids. Consistently, both SLC22A5/OCTN2 and SLC38A2/SNAT2 are induced during the differentiation of monocytes to macrophages at gene and protein levels. Elucidation of GM-CSF signaling demonstrates that the cytokine causes the activation of mTOR kinase, leading to the phosphorylation and activation of STAT3, which, in turn, is responsible for OCTN2 transcription. SLC22A5/OCTN2 therefore emerges as a novel member of the set of genes markers of macrophage differentiation.

Glutamine and leucine are important mTORC1 modulators, although their roles are not precisely defined. In HepG2 and HeLa cells glutamine-free incubation lowers mTORC1 activity, although cell leucine is not decreased. mTORC1 activity, suppressed by amino acid-free incubation, is completely rescued only if essential amino acids (EAA) and glutamine are simultaneously restored, although cell leucine is higher in the absence than in the presence of glutamine. Thus, glutamine stimulates mTORC1 independent of cell leucine, suggesting the existence of two distinct stimulatory signals from either glutamine or EAA.

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.

Carnitine plays a physiologically important role in the β-oxidation of fatty acids, facilitating the transport of long-chain fatty acids across the inner mitochondrial membrane. Distribution of carnitine within the body tissues is mainly performed by novel organic cation transporter (OCTN) family, including the isoforms OCTN1 (SLC22A4) and OCTN2 (SLC22A5) expressed in human. We performed here a characterization of carnitine transport in human airway epithelial cells A549, Calu-3, NCl-H441, and BEAS-2B, by means of an integrated approach combining data of mRNA/protein expression with the kinetic and inhibition analyses of L-[(3)H]carnitine transport. Carnitine uptake was strictly Na(+)-dependent in all cell models. In A549 and BEAS-2B cells, carnitine uptake was mediated by one high-affinity component (Km2 μM) identifiable with OCTN2. In both these cell models, indeed, carnitine uptake was maximally inhibited by betaine and strongly reduced by SLC22A5/OCTN2 silencing. Conversely, Calu-3 and NCl-H441 exhibited both a high (Km~20 μM) and a low affinity (Km1 mM) transport component. While the high affinity component is identifiable with OCTN2, the low affinity uptake is mediated by ATB(0,+), a Na(+), and Cl(-)-coupled transport system for neutral and cationic amino acids, as demonstrated by the inhibition by leucine and arginine, as well as by SLC6A14/ATB(0,+) silencing. The presence of this transporter leads to a massive accumulation of carnitine inside the cells and may be of peculiar relevance in pathologic conditions of carnitine deficiency, such as those associated to OCTN2 defects.

We report here that chlorpromazine, a first generation antipsychotic drug, inhibits anionic amino acid transport mediated by system X(-) (AG) (EAAT transporters) in cultured human fibroblasts. With 30 microM chlorpromazine, transport inhibition is detectable after 3 h of treatment, maximal after 48 h (60%), and referable to a decrease in V(max). Chlorpromazine effect is not dependent upon changes of membrane potential and is selective for system X(-) (AG) since transport systems A and y(+) are not affected. Among antipsychotic drugs, the inhibitory effect of chlorpromazine is shared by two dibenzodiazepines, clozapine and olanzapine, while other compounds, such as risperidon, zuclopentixol, sertindol and haloperidol, are not effective. Transport inhibition by clozapine and olanzapine, but not by chlorpromazine, is reversible, suggesting that the mechanisms involved are distinct. These results indicate that a subset of antipsychotic drugs inhibits EAAT transporters in non-nervous tissues and prompt further investigation on possible alterations of glutamate transport in peripheral tissues of schizophrenic patients.

Sodium-dependent neutral amino acid transporter-2 (SNAT2), the ubiquitous member of SLC38 family, accounts for the activity of transport system A for neutral amino acids in most mammalian tissues. As the transport process performed by SNAT2 is highly energized, system A substrates, such as glutamine, glycine, proline and alanine, reach high transmembrane gradients and constitute major components of the intracellular amino acid pool. Moreover, through a complex array of exchange fluxes, involving other amino acid transporters, and of metabolic reactions, such as the synthesis of glutamate from glutamine, SNAT2 activity influences the cell content of most amino acids, thus determining the overall size and the composition of the intracellular amino acid pool. As amino acids represent a large fraction of cell organic osmolytes, changes of SNAT2 activity are followed by modifications in both cell amino acids and cell volume. This mechanism is utilized by many cell types to perform an effective regulatory volume increase (RVI) upon hypertonic exposure. Under these conditions, the expression of SNAT2 gene is induced and newly synthesized SNAT2 proteins are preferentially targeted to the cell membrane, leading to a significant increase of system A transport Vmax. In cultured human fibroblasts incubated under hypertonic conditions, the specific silencing of SNAT2 expression, obtained with anti-SNAT2 siRNAs, prevents the increase in system A transport activity, hinders the expansion of intracellular amino acid pool, and significantly delays cell volume recovery. These results demonstrate the pivotal role played by SNAT2 induction in the short-term hypertonic RVI and suggest that neutral amino acids behave as compatible osmolytes in hypertonically stressed cells.

The activity and the membrane expression of EAAT3 glutamate transporter are stimulated upon PKC activation by phorbol esters in C6 rat glioma cells. To investigate the role of cytoskeleton in these effects, we have employed actin-perturbing toxins and found that the perturbation of actin cytoskeleton inhibits basal but not phorbol-stimulated EAAT3 activity and membrane trafficking. In the absence of phorbols, latrunculin A, a toxin that disassembles actin cytoskeleton, produced a rapid inhibition of EAAT3 activity, due to a decrease in transport Vmax. The inhibitory effect was fully reversible and was not detected for other sodium dependent transport systems for amino acids. However, latrunculin did not prevent the increase in transport caused by phorbol esters and, moreover, cells pre-treated with phorbols were resistant to the inhibitory effect of the toxin on EAAT3 activity. Biotinylation experiments indicated that the inhibitory effect of latrunculin was attributable to a decreased expression of the carrier on the membrane, while the toxin did not suppress the PKC-dependent increase in EAAT3 membrane abundance. Latrunculin A effects on EAAT3 were shared by cytochalasin D, a toxin that disorganizes actin filaments with a distinct mechanism of action. On the contrary, a small, but significant, increase of EAAT3 activity was observed upon incubation with jasplakinolide, a drug that stabilizes actin microfilaments. Also jasplakinolide, however, did not hinder phorbol-dependent stimulation of aspartate transport. Colchicine, a toxin that disrupts microtubules, also lowered EAAT3 activity without preventing transport stimulation by phorbols, while microtubule stabilization by paclitaxel led to an increase in aspartate transport. It is concluded that, in C6 cells, the PKC-mediated stimulatory effects on EAAT3 are cytoskeleton-independent, while in the absence of phorbols, the transporter is partially inhibited by the disorganization of either actin microfilaments or microtubules. These results suggest that EAAT3 trafficking in C6 cells involves different pools of transporters. # 2005 Elsevier Ltd. All rights reserved.

In cultured human fibroblasts incubated under hypertonic conditions, the stimulation of system A for neutral amino acid transport, associated to the increased expression of the mRNA for SNAT2 transporter, leads to an expanded intracellular amino acid pool and to the recovery of cell volume. A protein of nearly 60 kDa, recognized by an antiserum against SNAT2, is increased both in the pool of biotinylated membrane proteins and in the total cell lysate of hypertonically stressed cells. The increased level of SNAT2 transporters in hypertonically stressed cells is confirmed by immunocytochemistry. DRB, an inhibitor of transcription, substantially inhibits the increase of SNAT2 proteins on the plasma membrane, completely suppresses the stimulation of system A transport activity, and markedly delays the cell volume recovery observed during the hypertonic treatment. On the contrary, if the transport activity of system A is adaptively increased by amino acid starvation in the presence of DRB, the increase of SNAT2 transporters on the plasma membrane is still clearly detectable and the transport change only partially inhibited. It is concluded that the synthesis of new SNAT2 transporters is essential for the hypertonic stimulation of transport system A, but accounts only in part for the adaptive increase of the system.

In human saphenous vein endothelial cells (HSVECs), tumor necrosis factor-alpha (TNFalpha) and bacterial lipopolysaccharide (LPS), but neither interferon gamma (IFNgamma) nor interleukin 1beta (IL-1beta), stimulate arginine transport. The effects of TNFalpha and LPS are due solely to the enhancement of system y+ activity, whereas system y+L is substantially unaffected. TNFalpha causes an increased expression of SLC7A2/CAT-2B gene while SLC7A1/CAT-1 expression is not altered by the cytokine. The suppression of PKC-dependent transduction pathways, obtained with the inhibitor chelerytrhine, the inhibitor peptide of PKCzeta isoform, or chronic exposure to phorbol esters, does not prevent TNFalpha effect on arginine transport. Likewise, ERK, JNK, and p38 MAP kinases are not involved in the cytokine effect, since arginine transport stimulation is unaffected by their specific inhibitors. On the contrary, inhibitors of NF-kappaB pathway hinder the increase in CAT2B mRNA and the stimulation of arginine uptake. These results indicate that in human endothelial cells the activation of NF-kappaB pathway mediates the TNFalpha effects on arginine transport.

When grown on permeable supports, pancreatic duct adenocarcinoma CAPAN-1 cells establish very high values of transepithelial resistance (TER). The addition of ethanol produced a dose-related, reversible drop in the TER of these cells, ranging from 15% (with 1% ethanol) to 65% (with 10% ethanol). The ethanol effect was rapid and reversible. The resistance decrease was associated with an increase in monolayer permeability to mannitol. No significant decrease in cell ATP was detected for ethanol concentrations lower than 7%. Confocal vertical sections of calcein-loaded monolayers of CAPAN-1 cells, grown on plasticware, showed a progressive deflation of domes detectable after 5 min of treatment with 2% ethanol. Incubation in an ethanol-free medium caused a progressive dome restoration. Immunocytochemical analysis of ethanol-treated cells indicated that ZO-1 and occludin exhibited clear cut distribution changes while the perijunctional actin pattern was slightly modified. Electron microscopy showed that a discrete intercellular space was detectable between adjacent ethanol-treated cells but not between control cells. These data indicate that ethanol is a tight junction barrier opener in pancreatic duct cells.

Background . We evaluated the effects of standard preservation solutions on cultured human greater saphenous vein endothelial cells. Methods . Endothelial cells (eight strains) were preincubated for 6 or 24 hours at 4°C in Celsior, Euro-Collins, St. Thomas Hospital II, and University of Wisconsin solutions, reincubated in warm oxygenated culture medium 199, and observed up to 48 hours. Culture viability was assessed through cell counting and confocal microscopy of calcein loaded cells. Results . Incubation in both Euro-Collins and St. Thomas, but not in Celsior or University of Wisconsin solutions, caused significant cells losses and diffuse morphological damages characterized by solution-specific distinctive alterations. Injury caused by 6-hour, but not by 24-hour treatment, was reversible. Conclusions . The incubation with Celsior and University of Wisconsin solutions substantially preserved endothelial viability and proliferative capability. Conversely, a prolonged incubation in either Euro-Collins or St. Thomas solutions caused severe and potentially irreversible damage referable to the induction of, respectively, apoptotic or necrotic changes.

Many epithelial cells cultured on plastic ware form domes, fluid-filled localized raisings of the cell monolayer. Domes are due to active vectorial ion transport and their presence demonstrates the maintenance of a differentiated polarized phenotype and of tight junctional complexes. Through a confocal laser microscope equipped with a special flow chamber, intact domes were evaluated in real time for prolonged experimental periods. Both in CAPAN-1 pancreatic duct adenocarcinoma cells and in renal tubular LLC-PK1 cells, vertical sections of calcein-loaded cultures provided a clear visualization of dome outlines during the slow deflation induced by specific agonists (respectively, 1 µM secretin or 10 µM vasopressin). Section series of calcein-loaded domes were used for three-dimensional reconstructions. In CAPAN-1 cultures, cell depolarization induced by secretin was detected with the potentiometric dye bis-oxonol. In the same cells pyranine, a fluid phase marker that is cell impermeant, visualized dome compartment and paracellular pathways, also providing an evaluation of dome fluid pH. Confocal laser scanning microscopy of domes represents a convenient device for the functional assessment of living epithelial cells.

In lysinuric protein intolerance (LPI), impaired transport of cationic amino acids in kidney and intestine is due to mutations of the SLC7A7 gene. To assess the functional consequences of the LPI defect in nonepithelial cells, we have characterized cationic amino acid (CAA) transport in human fibroblasts obtained from LPI patients and a normal subject. In both cell types the bidirectional fluxes of arginine are due to the additive contributions of two Na+-independent, transstimulated transport systems. One of these mechanisms, inhibited by N-ethylmaleimide (NEM) and sensitive to the membrane potential, is identifiable with system y+. The NEM- and potential-insensitive component, suppressed by l-leucine only in the presence of Na+, is mostly due to the activity of system y+L. The inward and outward activities of the two systems are comparable in control and LPI fibroblasts. Both cell types express SLC7A1 (CAT1) and SLC7A2 (CAT2B and CAT2A) as well as SLC7A6 (y+LAT2) and SLC7A7 (y+LAT1). We conclude that LPI fibroblasts exhibit normal CAA transport through system y+L, probably referable to the activity of SLC7A6/y+LAT2.

Although morphological criteria for apoptosis are in general reliable, no systematic comparison of the techniques employed thus far has yet been performed. In this study, using confocal laser microscopy, we compared the performance of annexin V-FITC and calcein-AM for early detection of apoptosis in living adherent cells. Experiments were carried out on two distinct cell lines, PC 12 and NIH3T3, endowed with different shape and adhesion properties. The apoptotic process was followed for a prolonged period in the same cells of a predetermined field by means of a special flow chamber. Our results show that both probes allowed the detection of apoptotic cells in either cell line. However, some cells that clearly exhibited apoptotic changes on calcein visualization were annexin-negative. In NIH3T3 cells, annexin negativity of apoptotic cells was correlated with the preservation of cell shape and adhesion properties. These findings show that, at least in PC12 and NIH3T3 cells, annexin might be less sensitive than calcein-AM for early apoptosis detection and, for NIH3T3 cells, suggest that phosphatidilserine exposure is in some way linked to changes in cell shape and/or adhesion to culture substrate. (J Histochem Cytochem 46:895-900, 1998)

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).