Molecular and Functional Study of Transient Receptor Potential Vanilloid 1-4 at the Rat and Human Blood-Brain Barrier Reveals Interspecies Differences.

Huilong Luo,Nicolas Perrière,Véronique Cochois,Fabienne Glacial,Salvatore Cisternino,Catarina Chaves,Stéphanie Chasseigneaux,Xavier Declèves,Maria Smirnova,Bruno Saubamea

doi:10.3389/fcell.2020.578514

Abstract

Transient receptor potential vanilloid 1-4 (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 [rat 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 human cerebral microvessel endothelial cells D3 cells (hCMEC/D3) and in primary cultures of BMEC isolated from human adult and children brain resections [human brain microvascular endothelial cells (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 specific 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.

Highlights

During the last decade, the transient receptor potential (TRP) channels were identified as promising pharmacological targets and some drug candidates targeting TRP have even already entered clinical trials or been approved as drugs for treating neuropathic pain, epilepsia or heart failure (Moran, 2018)
No significant difference was observed for the gene expression of Trpv1 and Trpv3 between brain microvessels and rPBMEC (Figure 1A), while a significant decrease of Trpv2 and Trpv 4 gene expression was observed in rPBMEC compared with that in brain microvessels (Figure 1A, p < 0.01, p < 0.05 for Trpv2 and Trpv4, respectively, rat microvessels vs. rPBMEC)
Our present study shows that TRPV1, TRPV2 and TRPV4 are expressed in hPBMECs

Summary

Introduction

The transient receptor potential (TRP) channels were identified as promising pharmacological targets and some drug candidates targeting TRP have even already entered clinical trials or been approved as drugs for treating neuropathic pain, epilepsia or heart failure (Moran, 2018). The main knowledge of TRP functions and agonists come from their expression in sensory electrically excitable cells such as neurons and their role in the nociception and pain (Mickle et al, 2015). More studies have been done and evidenced the TRP expression and their ability in modulating the functionalities of non-excitable cells such as endothelial cells using various chemical agonists and antagonists, including a requirement in vasorelaxation and vascular permeability (Kassmann et al, 2013; Grace et al, 2014; Ambrus et al, 2017). TRPV5 and TRPV6 are mainly expressed in intestinal and renal epithelia (Peng et al, 2018) while transient receptor potential vanilloid 1-4 (TRPV1-4) are mostly expressed in diverse cell types of the CNS, exocrine organs, skin, eye, lung, heart, and blood vessels (Steinritz et al, 2018)

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