0000000000542913
AUTHOR
Valeria Dall'asta
SNAT2 silencing prevents the osmotic induction of transport system A and hinders cell recovery from hypertonic stress.
AbstractUnder 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.
INFγ stimulates arginine transport through system y+L in human monocytes
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.
In Lysinuric Protein Intolerance system y+L activity is defective in monocytes and in GM-CSF-differentiated macrophages
Abstract 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 a…
In human endothelial cells rapamycin causes mTORC2 inhibition and impairs cell viability and function.
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 impair…
The synthesis of SNAT2 transporters is required for the hypertonic stimulation of system A transport activity
AbstractIn 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 …
Gliadin activates arginase pathway in RAW264.7 cells and in human monocytes
AbstractCeliac 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 …
Gliadin-mediated production of polyamines by RAW264.7 macrophages modulates intestinal epithelial permeability in vitro
AbstractCeliac 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 permeabilit…
Rapamycin stimulates arginine influx through CAT2 transporters in human endothelial cells
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 stimulation of arginine transport by TNFα in human endothelial cells depends on NF-κB activation
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 TNFalp…