Abstract
Purpose:
The release of transmitters by secretory lysosomes represents a novel method of cell signaling. Here we asked whether lysosomal stores of ATP were released after TLR3 stimulation in optic nerve head astrocytes and RPE cells.
Methods:
Experiments were performed on mouse RPE cells, ARPE-19 cells, and rat optic nerve head astrocytes. Released ATP was detected using the luciferase assay, acid phosphatase was measured with an Elisa, and TLR3-phosphorylation with a Western blot.
Results:
Stimulation of TLR3 with agonist poly(I:C) released ATP from astrocytes and RPE cells. Extracellular ATP levels peaked after 15-30 min poly(I:C) in astrocytes, but over an hour in RPE cells. Poly(I:C) led to TLR3 phosphorylation, while ATP release was triggered by 21-mer but not 16-mer siRNA. ATP release was blocked by vesicular transport inhibitor NEM, but not pannexin inhibitor carbenoxelone. Fluorescent MANT-ATP showed particulate staining which colocalized with lysosomal stains. Poly(I:C) released lysosomal enzyme acid phosphatase with ATP, implying lysosomal exocytosis upon TLR3 activation. Both ATP and acid phosphatase were released upon stimulation of cells with scrambled siRNA. Serum starvation, but not rapamycin, prevented the ATP release triggered by poly(I:C).
Conclusions:
Stimulation of TLR3 activates lysosomes in astrocytes and RPE cells. The resulting lysosomal exocytosis leads to ATP release into the surrounding extracellular space. This may identify lysosomal activation, exocytosis and release of lysosomal ATP as a widespread cellular response to double stranded RNA exposure and TLR3 stimulation. The role of this released ATP and other lysosomal constituents in the reaction to TLR3 stimulation remains to be determined.