Human fibroblast cells are an advantageous model to study the transport of amino acids across cell membranes, since one can control the environmental factors. A major problem in all earlier studies is the lack of precise and detailed knowledge regarding the expression and functionality of tyrosine transporters in human fibroblasts. This motivated us to perform a systematic functional characterization of the tyrosine transport in fibroblast cells with respect to the isoforms of system-L (LAT1, LAT2, LAT3, LAT4), which is the major transporter of tyrosine. Ten (n=10) fibroblast cell lines from healthy volunteers were included in the study. Uptake of L-[U-14C] tyrosine in fibroblasts was measured using the cluster tray method in the presence and absence of excess concentrations of various combinations of inhibitors. This study demonstrated that LAT1 is involved in 90% of total uptake of tyrosine and also around 51% of alanine. Not more than 10% can be accounted for by LAT2, LAT3 and LAT4 isoforms. LAT2 seems to be functionally weak in uptake of tyrosine while LAT3 and LAT4 contributed around 7%. 10% could be contributed by system-A (ATA2 isoform). Alanine consequently inhibited the tyrosine transport by up to 60%. Tyrosine transport through the LAT1 isoform has a higher affinity compared to system-L. In conclusion, the LAT1 isoform is the major transporter of tyrosine in human fibroblast cells. Competition between tyrosine and alanine for transport is shown to exist, probably between LAT1 and LAT2 isoforms. This study established fibroblast cells as a suitable experimental model for studying amino acid transport defects in humans.

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BackgroundThe catecholaminergic and serotonergic neurotransmitter systems are implicated in the pathophysiology of attention-deficit/hyperactivity disorder (ADHD). The amino acid tyrosine is the precursor for synthesis of the catecholamines dopamine and norepinephrine, while tryptophan is the precursor of serotonin. A disturbed transport of tyrosine, as well as other amino acids, has been found in a number of other psychiatric disorders, such as schizophrenia, bipolar disorder and autism, when using the fibroblast cell model. Hence, the aim of this study was to explore whether children with ADHD may have disturbed amino acid transport.MethodsFibroblast cells were cultured from skin biopsies obtained from 14 boys diagnosed with ADHD and from 13 matching boys without a diagnosis of a developmental disorder. Transport of the amino acids tyrosine, tryptophan and alanine across the cell membrane was measured by the cluster tray method. The kinetic parameters, maximal transport capacity (Vmax) and affinity constant (Km) were determined. Any difference between the two groups was analyzed by Student's unpaired t-test or the Mann Whitney U test.ResultsThe ADHD group had significantly decreased Vmax (p = 0.039) and Km (increased affinity) (p = 0.010) of tryptophan transport in comparison to controls. They also had a significantly higher Vmaxof alanine transport (p = 0.031), but the Km of alanine transport did not differ significantly. There were no significant differences in any of the kinetic parameters regarding tyrosine transport in fibroblasts for the ADHD group.ConclusionsTryptophan uses the same transport systems in both fibroblasts and at the blood brain barrier (BBB). Hence, a decreased transport capacity of tryptophan implies that less tryptophan is being transported across the BBB in the ADHD group. This could lead to deficient serotonin access in the brain that might cause disturbances in both the serotonergic and the catecholaminergic neurotransmitter systems, since these systems are highly interconnected. The physiological importance of an elevated transport capacity of alanine to the brain is not known to date.

Background/Aims: Increased nitric oxide (NO) production or inducible form of NO synthase activity have been documented in patients suffering from urinary tract infection (UTI), but the role of NO in this infection is unclear. We investigated whether NO can affect the host response in human renal epithelial cells by modulating IL-6 production and mRNA expression. Methods: The human renal epithelial cell line A498 was infected with a uropathogenic Escherichia coli (UPEC) strain and/or the NO donor DETA/NO. The IL-6 production and mRNA expression were evaluated by ELISA and real-time RT-PCR. IL-6 mRNA stability was evaluated by analyzing mRNA degradation by real-time RT-PCR. Results: DETA/NO caused a significant (p < 0.05) increase in IL-6 production. Inhibitors of p38 MAPK and ERK1/2 signaling, but not JNK, were shown to significantly suppress DETA/NO-induced IL-6 production. UPEC-induced IL-6 production was further increased (by 73 ± 23%, p < 0.05) in the presence of DETA/NO. The IL-6 mRNA expression increased 2.1 ± 0.17-fold in response to DETA/NO, while the UPEC-evoked increase was pronounced (20 ± 4.5-fold). A synergistic effect of DETA/NO on UPEC-induced IL-6 expression was found (33 ± 7.2-fold increase).The IL-6 mRNA stability studies showed that DETA/NO partially attenuated UPEC-induced degradation of IL-6 mRNA. Conclusions: NO was found to stimulate IL-6 in renal epithelial cells through p38 MAPK and ERK1/2 signaling pathways and also to increase IL-6 mRNA stability in UPEC-infected cells. This study proposes a new role for NO in the host response during UTI by modulating the transcription and production of the cytokine IL-6.

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