Hyperosmotic stress induces ATP release and changes in P2X7 receptor levels in human corneal and conjunctival epithelial cells

Abstract

Tear hyperosmolarity is a key event in dry eye. In this work, we analyzed whether hyperosmolar challenge induces ATP release on the ocular surface. Moreover, as extracellular ATP can activate P2X7 receptor, the changes in P2X7 protein levels and its involvement in pathological process triggered by hypertonic treatment were also examined. Highperformance liquid chromatography analysis revealed that ATP levels significantly increased in human corneal and conjunctival epithelial cells exposed to hyperosmotic challenge as well as in dry eye patients as compared to control subjects. A significant reduction in cell viability was detected after hyperosmolar treatment, indicating that the rise in ATP release was mainly due to cell lysis/death. Additionally, vesicular nucleotide transporter was identified in both cell lines and their protein expression was upregulated in hypertonic media. P2X7 receptor truncated form together with the full-length form was identified in both cell lines, and experiments using specific antagonist and agonist for P2X7 indicated that this receptor did not mediate cell death induced by hyperosmolar stress. In conclusion, hyperosmotic stress induces ATP release. Extracellular ATP can activate P2X7 receptor leading to cytotoxicity in many cells/tissues; however, this does not occur in human corneal and conjunctival epithelial cells. In these cells, the presence of P2X7 receptor truncated form together with the full-length form hinders a P2X7 apoptotic behavior on the ocular surface.

Blue light phototoxicity toward human corneal and conjunctival epithelial cells in basal and hyperosmolar conditions

“…In order to mimic in vitro dry eye conditions, HO stress was applied to these cells [38]. To make HO media, we added 69 mM of NaCl since greater values were reported to induce an important cell death [38,44] and would probably bias the impact of light phototoxicity. The measured osmolarity values were within the range of the hyperosmolarity commonly used in experimental settings (e.g.…”

Vesicular nucleotide transporter (VNUT): appearance of an actress on the stage of purinergic signaling

“…4) (Table 1). VNUT expression was also detected in the cultured human corneal limbal epithelial cells and human conjunctival epithelial cells, although the organelles containing VNUT were unidentified [82]. VNUT is also expressed in type II taste cells in taste buds [83], mouse esophageal keratinocytes [84], cat esophageal mucosa [85], glucagon-containing secretory granules in islet α and insulin granules of β cells [86,87], Schwann cells [88], human lung cancer A549 cells [89], human airway epithelial goblet Calu-3 cells [54], zymogen granules of mouse pancreatic acinar cells [90,91], GLP-1-containing vesicles of intestinal L cells [92], rat parotid glands [93], rat liver epithelium [94], biliary epithelial cells [95], platelets [96], odontoblasts [97], and bladder urothelium [98] (Fig.…”

Cough-provocation tests with hypertonic aerosols

“…ATP is a molecule with several potential functions, such as an energy source, a transmitter, a paracrine signalling molecule and an extracellular reporter of ischaemia, damage and inflammation [33]. It has been shown that human lens, retinal, corneal and conjunctival epithelial cells release ATP in response to hypertonicity [34,35]. Thus, it is possible that hypertonicity could activate airway C-fibre P2X3 receptors by stimulating ATP release from airway epithelial cells.…”