Lysosomal TRPML1 triggers global Ca signals and nitric oxide release in human cerebrovascular endothelial cells.

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.