Pretreatment Effect of Inflammatory Stimuli and Characteristics of Tryptophan Transport on Brain Capillary Endothelial (TR-BBB) and Motor Neuron Like (NSC-34) Cell Lines

Tryptophan plays a key role in several neurological and psychiatric disorders. In this study, we investigated the transport mechanisms of tryptophan in brain capillary endothelial (TR-BBB) cell lines and motor neuron-like (NSC-34) cell lines. The uptake of [3H]l-tryptophan was stereospecific, and concentration- and sodium-dependent in TR-BBB cell lines. Transporter inhibitors and several neuroprotective drugs inhibited [3H]l-tryptophan uptake by TR-BBB cell lines. Gabapentin and baclofen exerted a competitive inhibitory effect on [3H]l-tryptophan uptake. Additionally, l-tryptophan uptake was time- and concentration-dependent in both NSC-34 wild type (WT) and mutant type (MT) cell lines, with a lower transporter affinity and higher capacity in MT than in WT cell lines. Gene knockdown of LAT1 (l-type amino acid transporter 1) and CAT1 (cationic amino acid transporter 1) demonstrated that LAT1 is primarily involved in the transport of [3H]l-tryptophan in both TR-BBB and NSC-34 cell lines. In addition, tryptophan uptake was increased by TR-BBB cell lines but decreased by NSC-34 cell lines after pro-inflammatory cytokine pre-treatment. However, treatment with neuroprotective drugs ameliorated tryptophan uptake by NSC-34 cell lines after inflammatory cytokines pretreatment. The tryptophan transport system may provide a therapeutic target for treating or preventing neurodegenerative diseases.

Tryptophan Transport in Human Fibroblast Cells—A Functional Characterization

There are indications that serotonergic neurotransmission is disturbed in several psychiatric disorders. One explanation may be disturbed transport of tryptophan (precursor for serotonin synthesis) across cell membranes. Human fibroblast cells offer an advantageous model to study the transport of amino acids across cell membranes, since they are easy to propagate and the environmental factors can be controlled. The aim of this study was to functionally characterize tryptophan transport and to identify the main transporters of tryptophan in fibroblast cell lines from healthy controls. Tryptophan kinetic parameters ( Vmax and Km) at low and high concentrations were measured in fibroblasts using the cluster tray method. Uptake of 3H (5)-L-tryptophan at different concentrations in the presence and absence of excess concentrations of inhibitors or combinations of inhibitors of amino acid transporters were also measured. Tryptophan transport at high concentration (0.5 mM) had low affinity and high Vmax and the LAT1 isoform of system-L was responsible for approximately 40% of the total uptake of tryptophan. In comparison, tryptophan transport at low concentration (50 nM) had higher affinity, lower Vmax and approximately 80% of tryptophan uptake was transported by system-L with LAT1 as the major isoform. The uptake of tryptophan at the low concentration was mainly sodium (Na+) dependent, while uptake at high substrate concentration was mainly Na+ independent. A series of different transporter inhibitors had varying inhibitory effects on tryptophan uptake. This study indicates that tryptophan is transported by multiple transporters that are active at different substrate concentrations in human fibroblast cells. The tryptophan transport trough system-L was mainly facilitated by the LAT1 isoform, at both low and high substrate concentrations of tryptophan.