Molecular and Functional Study of Transient Receptor Potential Vanilloid 1-4 at the Rat and Human Blood–Brain Barrier Reveals Interspecies Differences
Huilong Luo, Bruno Saubaméa, Stéphanie Chasseigneaux, Véronique Cochois, Maria Smirnova, Fabienne Glacial, Nicolas Perrière, Catarina Chaves, Salvatore Cisternino, Xavier Declèves
Abstract
TRPV1-4 expression and functionality were investigated in brain microvessel endothelial cells (BMEC) forming the blood-brain barrier (BBB) from rat and human origins. In rat, Trpv1-4 were detected by qRT-PCR in the brain cortex, brain microvessels, and in primary cultures of brain microvessel endothelial cells (rPBMEC). A similar Trpv1-4 expression profile in isolated brain microvessels and rPBMEC was found with the following order: Trpv4 > Trpv2 > Trpv3 > Trpv1. In human, TRPV1-4 were detected in the BBB cell line hCMEC/D3 and in primary cultures of BMEC isolated from human adult and children brain resections (hPBMEC), showing a similar TRPV1-4 expression profile in both hCMEC/D3 cells and hPBMECs as follow: TRPV2 >> TRPV4 > TRPV1 > TRPV3. Western blotting and immunofluorescence experiments confirmed that TRPV2 and TRPV4 are the most expressed TRPV isoforms in hCMEC/D3 cells with a clear staining at the plasma membrane. A fluorescent dye Fluo-4 AM ester was applied to record intracellular Ca2+ levels. TRPV4 functional activity was demonstrated in mediating Ca2+ influx under stimulation with the specific agonist GSK1016790A (ranging from 3 to 1000 nM, EC50 of 16.2 ± 4.5 nM), which was inhibited by the specific TRPV4 antagonist, RN1734 (30 µM). In contrast, TRPV1 was slightly activated in hCMEC/D3 cells as shown by the weak Ca2+ influx induced by capsaicin at a high concentration (3 µM), a highly potent and specikfic TRPV1 agonist. Heat-induced Ca2+ influx was not altered by co-treatment with a selective potent TRPV1 antagonist capsazepine (20 µM), in agreement with the low expression of TRPV1 as assessed by qRT-PCR. Our present study reveals an interspecies difference between Rat and Human. Functional contributions of TRPV1-4 subtype expression were not identical in rat and human tissues reflective of BBB integrity. TRPV2 was predominant in the human whereas TRPV4 had a larger role in the rat. This interspecies difference from a gene expression point of view should be taken into consideration when modulators of TRPV2 or TRPV4 are investigated in rat models of brain disorders.