Effects of ATP and Its Analogues on (Ca2+)i Dynamics in the Rabbit Corneal Epithelium.

Abstract

Adenosine-5'-triphosphate (ATP) plays a pivotal role in various tissues as an extracellular transmitter. ATP released from nerve endings and/or damaged cells may elicit reactions in adjacent cells. To identify such reactions, we investigated the dynamics of the intracellular calcium ion concentrations ([Ca2+]i) in the rabbit corneal epithelium during ATP-stimulation. Intact epithelial sheets isolated from corneal tissue were loaded with Fura-2, and [Ca2+]i dynamics in each cell layer were analyzed using a digital imaging system (Argus 50/CA). Normal architecture was preserved, suggesting that functional integrity remained intact. Perfusion with HEPES-buffered Ringer's solution containing ATP (10 microM) and uridine-5'-triphosphate (UTP; 10 microM) caused a biphasic [Ca2+]i increase in the superficial layer that manifested itself as a rapid initial spike followed by a long-lasting plateau phase. Adenosine-5'-diphate (10 microM) elevated the [Ca2+]i level, but induced only the initial spike, which was smaller than those induced by ATP and UTP. Adenosine (10 microM) did not elicit any [Ca2+]i changes in the epithelial cells. Suramin (10 microM; a P2 receptor antagonist) blocked the ATP-induced [Ca2+]i increase, whereas the P2X receptor agonists, alpha, beta-methylene ATP (10 microM), 2-methyl-thio ATP (10 microM) and Benzoylbenzoyl ATP (10 microM), did not elicit any increases in [Ca2+]i. In the basal cell layer, ATP-induced [Ca2+]i dynamics were biphasic, while oscillatory fluctuations of [Ca2+]i were induced in the wing cells of the mid layer of the corneal epithelium by ATP stimulation. Ca2+ oscillations were sometimes synchronized among adjacent wing cells, but these waves did not propagate to other cell layers. These results suggest that extracellular ATP elicits a [Ca2+]i increase mainly via P2Y receptors. In addition, synchronized Ca2+ oscillation in the wing cell layer indicates that intracellular events may spread to neighboring cells within the layer.

P2Y receptors play a critical role in epithelial cell communication and migration

“…2), since ligand binding to a receptor expressed on the cellsurface is required for a response to occur. Furthermore, the Ca 2þ imaging results for the corneal epithelial cell line corresponded with those for primary epithelial cells isolated from rabbit corneas [Kimura et al, 1999] (Fig. 2B,C).…”

Wound-induced ATP release and EGF receptor activation in epithelial cells

“…Furthermore, our data showed that ADP induces low-level HB-EGF shedding and has no effect on wound closure, a result consistent with a recent report (Mediero et al, 2006), suggesting that ATP, but not ADP, serves as one of the initial signals to trigger wound response in HCE cells. Previous studies, using various inhibitors, showed that P2Y but not P2X receptors are responsible for ATP-induced Ca 2+ dynamics in human and rabbit corneal epithelium (Kimura et al, 1999;Klepeis et al, 2004). To assess the involvement of extracellular ATP in epithelial wound healing, we used two purinoceptor inhibitors suramin and RB2 to determine the effects of P2Y blockade on HCE cell wound healing.…”

Functional Expression of P2Y Receptors in WERI-Rb1 Retinoblastoma Cells

“…P2Y2 receptors have been identified in a wide variety of ocular cell types, particularly, in retina [26], retinal pigment epithelium [27][28][29], cornea [30,31], conjunctiva [32], lens [33], and ciliary epithelia [9,34]. P2Y2 receptor signaling and its function are apparent in the eye [35], and regulates multiple cellular functions in ocular physiology by calcium mobilization [9] including ion transport and fluid absorption [27,29], and mucin discharge [36].…”