Luo Huilong, Saubamea Bruno, Chasseigneaux Stéphanie, Cochois Véronique, Smirnova Maria, Glacial Fabienne, Perrière Nicolas, Chaves Catarina, Cisternino Salvatore, Declèves Xavier
Faculté de Pharmacie, Inserm, UMRS-1144, Optimisation Thérapeutique en Neuropsychopharmacologie, Université de Paris, Paris, France.
Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI, United States.
Front Cell Dev Biol. 2020 Nov 11;8:578514. doi: 10.3389/fcell.2020.578514. eCollection 2020.
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 expression profile in isolated brain microvessels and rPBMEC was found with the following order: > > > . 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 expression profile in both hCMEC/D3 cells and hPBMECs as follow: > > > > . 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 Ca levels. TRPV4 functional activity was demonstrated in mediating Ca influx under stimulation with the specific agonist GSK1016790A (ranging from 3 to 1000 nM, EC 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 Ca influx induced by capsaicin at a high concentration (3 μM), a highly potent and specific TRPV1 agonist. Heat-induced Ca influx was not altered by co-treatment with a selective potent TRPV1 antagonist capsazepine (20 μM), in agreement with the low expression of 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.
在来自大鼠和人类的形成血脑屏障（BBB）的脑微血管内皮细胞（BMEC）中，对瞬时受体电位香草酸亚型1 - 4（TRPV1 - 4）的表达和功能进行了研究。在大鼠中，通过qRT - PCR在大脑皮层、脑微血管以及脑微血管内皮细胞原代培养物[大鼠脑微血管内皮细胞（rPBMEC）]中检测到Trpv1 - 4。在分离的脑微血管和rPBMEC中发现了类似的表达谱，顺序如下：> > > 。在人类中，在血脑屏障细胞系人脑微血管内皮细胞D3细胞（hCMEC/D3）以及从成人和儿童脑切除术中分离的BMEC原代培养物[人脑微血管内皮细胞（hPBMEC）]中检测到TRPV1 - 4，在hCMEC/D3细胞和hPBMEC中显示出类似的表达谱，如下：> > > > 。蛋白质印迹和免疫荧光实验证实，TRPV2和TRPV4是hCMEC/D3细胞中表达最多的TRPV亚型，在质膜处有明显染色。应用荧光染料Fluo - 4 AM酯来记录细胞内钙水平。在特异性激动剂GSK1016790A（浓度范围为3至1000 nM，EC为16.2±4.5 nM）刺激下，TRPV4的功能活性表现为介导钙内流，这被特异性TRPV4拮抗剂RN1734（30μM）抑制。相比之下，在hCMEC/D3细胞中TRPV1略有激活，如高浓度（3μM）辣椒素诱导的微弱钙内流所示，辣椒素是一种高效且特异性的TRPV1激动剂。与qRT - PCR评估的低表达一致（此处表述有误，推测是指TRPV1低表达），与选择性强效TRPV1拮抗剂辣椒平（20μM）共同处理并未改变热诱导的钙内流。我们目前的研究揭示了大鼠和人类之间的种间差异。TRPV1 - 4亚型表达的功能贡献在反映血脑屏障完整性的大鼠和人类组织中并不相同。TRPV2在人类中占主导地位，而TRPV4在大鼠中作用更大。在脑疾病大鼠模型中研究TRPV2或TRPV4调节剂时，应从基因表达的角度考虑这种种间差异。
