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H. Yang, R. Pun, H. Liu, W.W. Kao, P.S. Reinach; Injury–Induced Purinergic Receptor Stimulation Elicits in–vivo Calcium Transients Across the Mouse Corneal Epithelium . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2902.
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© ARVO (1962-2015); The Authors (2016-present)
To characterize vivo in the mouse corneal epithelium, injury–induced calcium transients and changes in ion channel activity. The involvement of purinergic receptors in eliciting calcium signaling was also determined since in–vitro injury–induced calcium transients were shown to be dependent on purinergic receptor stimulation (Klepeis, et al, J Cell Science 2001).
Corneal epithelium debridement (2 mm in diameter) was created in experimental mice under anesthetization with an Alger® brush. Two different types of protocols were used to characterize calcium signaling: 1) with the in vivo protocol, 30' after Calcium Green 1 AM (5 µM) injection into the conjunctiva, injury–induced calcium transients were recorded using video imaging on the stage of a Zeiss upright microscope. Alternatively, an in vitro protocol was used: 2) after loading with fura2–AM (10 µM), ATP (100 µM)–induced calcium signaling was measured at different time intervals (1–6 h and 12–24 h) following wounding in the isolated cornea. Ion channel activity was recorded using the on–cell attached patch clamp configuration.
Transient increases in calcium spread immediately outwards from the site of injury, which persisted for 2–3 min followed by gradual declines towards baseline values over the next 60 min. In the 12–24 h injury group, the basal [Ca2+]i levels were about 2–fold above those corneas injured after 1–6 h (65± 4 versus 32± 4 nM, n=27, p<0.01). ATP–induced peak increases in [Ca2+]i in the corneas injury of 12–24 h were 38% higher than that 1–6 h of injury (125 ± 6 versus 91 ± 4 nM, n=27 p<0.01). In both groups, these increases were suppressed by 60% in the presence of 100 µM reactive blue 2 (RB2). Outward currents were observed in the 12–24 h injury group.
Calcium transients subsequent to injury may required for activation of intracellular signaling events eliciting increases in cell proliferation and migration that lead to healing. Such repetitive transients are in part attributable to purinergic receptor (P2Y) stimulation since: 1) either apyrase–induced ATP degradation or blocking P2Y with RB2 suppressed them; 2) ATP–induced transients could be elicited in the chronic–injury group and they were attenuated with RB2. The delay in activation of outward currents until at least 6 h after injury suggests that increases in ion channel activity and/or expression are dependent on upstream events activated by calcium signaling. The identity of the possible Ca2+–sensitive channels underlying the current increases awaits the outcome of additional studies.
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