Brunetti Valentina, Berra-Romani Roberto, Conca Filippo, Soda Teresa, Biella Gerardo Rosario, Gerbino Andrea, Moccia Francesco, Scarpellino Giorgia
Laboratory of General Physiology, Department of Biology and Biotechnology "L. Spallanzani", University of Pavia, Pavia, Italy.
Department of Biomedicine, School of Medicine, Benemérita Universidad Autónoma de Puebla, Puebla, Mexico.
Front Physiol. 2024 Jun 21;15:1426783. doi: 10.3389/fphys.2024.1426783. eCollection 2024.
Lysosomal Ca signaling is emerging as a crucial regulator of endothelial Ca dynamics. Ca release from the acidic vesicles in response to extracellular stimulation is usually promoted Two Pore Channels (TPCs) and is amplified by endoplasmic reticulum (ER)-embedded inositol-1,3,4-trisphosphate (InsP) receptors and ryanodine receptors. Emerging evidence suggests that sub-cellular Ca signals in vascular endothelial cells can also be generated by the Transient Receptor Potential Mucolipin 1 channel (TRPML1) channel, which controls vesicle trafficking, autophagy and gene expression. Herein, we adopted a multidisciplinary approach, including live cell imaging, pharmacological manipulation, and gene targeting, revealing that TRPML1 protein is expressed and triggers global Ca signals in the human brain microvascular endothelial cell line, hCMEC/D3. The direct stimulation of TRPML1 with both the synthetic agonist, ML-SA1, and the endogenous ligand phosphatidylinositol 3,5-bisphosphate (PI(3,5)P) induced a significant increase in [Ca] that was reduced by pharmacological blockade and genetic silencing of TRPML1. In addition, TRPML1-mediated lysosomal Ca release was sustained both by lysosomal Ca release and ER Ca- release through inositol-1,4,5-trisphophate receptors and store-operated Ca entry. Notably, interfering with TRPML1-mediated lysosomal Ca mobilization led to a decrease in the free ER Ca concentration. Imaging of DAF-FM fluorescence revealed that TRPML1 stimulation could also induce a significant Ca-dependent increase in nitric oxide concentration. Finally, the pharmacological and genetic blockade of TRPML1 impaired ATP-induced intracellular Ca release and NO production. These findings, therefore, shed novel light on the mechanisms whereby the lysosomal Ca store can shape endothelial Ca signaling and Ca-dependent functions in vascular endothelial cells.
溶酶体钙信号正逐渐成为内皮细胞钙动力学的关键调节因子。响应细胞外刺激时，酸性囊泡中的钙释放通常由双孔通道（TPCs）促进，并通过内质网（ER）嵌入的肌醇-1,3,4-三磷酸（InsP）受体和兰尼碱受体进行放大。新出现的证据表明，血管内皮细胞中的亚细胞钙信号也可由瞬时受体电位黏脂蛋白1通道（TRPML1）产生，该通道控制囊泡运输、自噬和基因表达。在此，我们采用了多学科方法，包括活细胞成像、药理学操作和基因靶向，揭示TRPML1蛋白在人脑微血管内皮细胞系hCMEC/D3中表达并触发全局钙信号。用合成激动剂ML-SA1和内源性配体磷脂酰肌醇3,5-二磷酸（PI(3,5)P）直接刺激TRPML1，可诱导[Ca]显著增加，而TRPML1的药理学阻断和基因沉默可降低该增加。此外，TRPML1介导的溶酶体钙释放通过肌醇-1,4,5-三磷酸受体介导的溶酶体钙释放和内质网钙释放以及储存性钙内流得以持续。值得注意的是，干扰TRPML1介导的溶酶体钙动员导致内质网游离钙浓度降低。DAF-FM荧光成像显示，TRPML1刺激还可诱导一氧化氮浓度显著的钙依赖性增加。最后，TRPML1的药理学和基因阻断损害了ATP诱导的细胞内钙释放和一氧化氮生成。因此，这些发现为溶酶体钙库塑造血管内皮细胞中内皮钙信号和钙依赖性功能的机制提供了新的见解。
