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J. Chow, P. Liton, F. Wong, P. Gonzalez; Alteration of P2Y Receptor–Mediated Calcium Response in Senescent Trabecular Meshwork Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5157.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: P2Y receptor–mediated calcium homeostasis is known to affect cell volume and shape in a variety of systems. P2Y receptors have been found in trabecular meshwork (TM) cells, where cell volume regulation could potentially play an important role in the modulation of aqueous humor outflow resistance. We set out to determine which P2Y receptor subtypes are involved in TM calcium regulation and to characterize how P2Y receptor–mediated calcium responses might be altered in stress–induced senescent TM cells. Methods: Porcine TM cells were plated on gelatin–coated coverslips and bathed in normal calcium Ringer’s solution. The cells were loaded with the fluorescent calcium indicator, fluo–4 (5µM), for one hour. Agonists of P2Y1 (ADP) and P2Y2/P2Y4 (ATP, UTP) receptors at 10 or 100µM 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). Calcium responses were compared in TM cells after incubation for two weeks in 5% O2 (physiological conditions) versus in 20% O2 (oxidative stress). Senescence was characterized by the presence of senescence–associated beta–galactosidase (sa–beta–gal) and lipofuscin autofluorescence, both quantified by flow cytometry. Results: 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 10µM ADP, 2.59 ± 0.33 (n=6) for 100µM ADP, 1.21 ± 0.64 (n=12) for 10µM UTP, 3.22 ± 2.0 (n=12) for 100µM UTP, 0.88 ± 0.40 (n=9) for 10µM ATP, and 1.37 ± 0.61 (n=25) for 100µM ATP. In oxidative stress–induced senescent cells, senescence markers sa–beta–gal and lipofuscin autofluorescence were elevated 4–fold and 12–fold, respectively. Compared to cells incubated in physiological conditions, the average calcium response in senescent cells decreased by 30% at 10µM UTP (Wilcoxon Rank–Sum test P value 0.024) and 29% at 100µM UTP (Wilcoxon Rank–Sum test P value<0.001). Conclusions: The concentration–dependent, agonist–induced rise in intracellular calcium supported the presence of functional P2Y1 and P2Y2/P2Y4 receptors in porcine TM cells. Compared to cells incubated in physiological conditions, the decrease in UTP–induced rise in intracellular calcium of senescent cells was statistically significant, suggesting that these cells may have altered calcium homeostasis. This change might contribute to the pathophysiology of glaucoma.
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