Effect of cellular senescence on the P2Y-receptor mediated calcium response in trabecular meshwork cells

PURPOSE: The objectives of this study were to evaluate the potential release of ATP that is mediated by mechanical stress on trabecular meshwork (TM) cells, to identify the specific P2Y receptors mediating the ATP response, and to determine whether cellular senescence might interfere with the P2Y receptor-mediated calcium response, thus contributing to the loss of physiologic TM function in aging and primary open angle glaucoma (POAG). METHODS: Experiments were conducted using primary cultures of porcine TM cells. Cyclic mechanical stretch (10% stretching/second) was generated using the Flexcell system. ATP release and ectoATPase activity induced by mechanical stress were measured using a luciferin/luciferase assay. Replicative senescence was induced by passing the cells 18 times at a 1:2 split ratio and confirmed by the presence of senescence-associated β-galactosidase (sa-β-gal) and autofluorescence. For calcium imaging, cells were plated on gelatin-coated coverslips, bathed in calcium Ringer's solution, and loaded with fluo-4 (5 μM) for 1 h. Agonists of P2Y1 (ADP) and P2Y2/P2Y4 (ATP, UTP) receptors at 10 μM or 100 μM concentrations were added to the bathing medium. Relative changes in cytosolic calcium concentration as a function of time were measured by fluorescent microscopy and reported as peak amplitudes of fluo-4 fluorescence normalized to baseline values (ΔF/Fo). RESULTS: Mechanical stress induced an increase in ATP release from TM cells (258%±23% at 15 min, 188%±11% at 30 min, and 900%±203% at 1 h; p<0.017, n=4) as well as an increase in ectoATPase activity present in the extracellular media during the first 15 min of stress (57%±15%, p=0.011, n=4). The P2Y receptor agonists listed above induced a concentration-dependent rise in intracellular calcium in the TM cells. The peak amplitude, ΔF/Fo, was 1.07±0.12 (n=3) for 10 μM ADP, 2.59±0.33 (n=6) for 100 μM ADP, 1.21±0.64 (n=12) for 10 μM UTP, 3.22±2.0 (n=12) for 100 μM UTP, 0.88±0.40 (n=9) for 10 μM ATP, and 1.37±0.61 (n=25) for 100 μM ATP. Cells at passage 18 showed significantly lower levels of intracellular calcium induced by ATP (36%), UTP (34%), and ADP (52%) compared to cells at passage 2, independent from any changes in P2Y receptor changes in expression. CONCLUSIONS: The ability to release ATP in response to mechanical stress and the presence of functional P2Y receptors in TM cells suggest a novel mechanism by which TM cells could sense and respond to changes in intraocular pressure (IOP). In addition, the decrease in P2Y receptor-mediated calcium responses observed in senescent TM cells suggests that the disregulation of calcium homeostasis in senescence may contribute to the alterations of the TM in aging and POAG.