Kynurenine pathway metabolism in human blood-brain-barrier cells: implications for immune tolerance and neurotoxicity.

The catabolic pathway of l-tryptophan (l-trp), known as the kynurenine pathway (KP), has been implicated in the pathogenesis of a wide range of brain diseases through its ability to lead to immune tolerance and neurotoxicity. As endothelial cells (ECs) and pericytes of the blood-brain-barrier (BBB) are among the first brain-associated cells that a blood-borne pathogen would encounter, we sought to determine their expression of the KP. Using RT-PCR and HPLC/GC-MS, we show that BBB ECs and pericytes constitutively express components of the KP. BBB ECs constitutively synthesized kynurenic acid, and after immune activation, kynurenine (KYN), which is secreted basolaterally. BBB pericytes produced small amounts of picolinic acid and after immune activation, KYN. These results have significant implications for the pathogenesis of inflammatory brain diseases in general, particularly human immunodeficiency virus (HIV)-related brain disease. Kynurenine pathway activation at the BBB results in local immune tolerance and neurotoxicity: the basolateral secretion of excess KYN can be further metabolized by perivascular macrophages and microglia with synthesis of quinolinic acid. The results point to a mechanism whereby a systemic inflammatory signal can be transduced across an intact BBB to cause local neurotoxicity.

Lysosomal traffic of liganded endothelin B receptor.

The endothelin B receptor (ETB) is an endothelial cell receptor found in caveolae. Studies with GFP-tagged ETB have suggested that the protein is constitutively endocytosed and targeted to lysosomes where it is rapidly degraded. We report that iodinated endothelin-1 ligand (ET-1) is taken up by cells transfected with ETB and remains undegraded for at least 17 h. Analysis of the intracellular traffic of endocytosed ET-1 on isotonic Ficoll gradients shows that it is rapidly internalised to lysosomes by a chloroquine sensitive and cholesterol dependent pathway. Low-temperature nonreducing SDS gels show that the ET-1 initially binds to full-length GFP-tagged ETB, which is rapidly clipped at the amino-terminus and is then stable for at least 6 h. Analysis of GFP tagged ETB on reducing SDS gels shows that it is proteolytically cleaved with a half time of approximately 3 h. However, nonreducing gels show that the receptor is virtually intact, suffering only a similar cleavage to the liganded receptor. We conclude that the ETB receptor shows remarkable stability in lysosomes, held together by disulfide bonds, and maintaining ligand binding for long periods of time.