Purinergic P2X Receptors Are Present and Functional in Conjunctival Goblet Cells

Abstract

Purpose : P2X receptors are activated by nucleotides such as ATP. Upon activation, the P2XRs become permeable to calcium which is a potent stimulator of mucin secretion in goblet cells. P2X7 receptors are capable of creating pores together with pannexin 1-3 hemichannels allowing flux of larger molecules and cell death in certain tissues. The purpose of this study was to investigate the presence and function of P2XRs in conjunctival goblet cells (CGCs).

Methods : Goblet cells were cultured from pieces of rat conjunctiva. Presence of P2X1-7 receptor and pannexin mRNA transcript expression and protein was determined by RT-PCR, western blotting (WB) analysis, and immunofluorescence (IF) microscopy. To study receptor function, CGCs were incubated with the ratiometric fluorescent dye Fura 2-AM, stimulated with ATP or the P2X7-specific ATP analogue BzATP, and intracellular [Ca²⁺] ([Ca²⁺]_i) measured. Extracellular Mg²⁺ was removed and the pH decreased to 6.8 before addition of ATP or BzATP to investigate if a P2X7- or P2X2-modulated increase in [Ca²⁺]_i occurred. [Ca²⁺]_i was also measured after CGCs were preincubated with the P2X7 antagonist A804598 and stimulated with ATP or BzATP.

Results : Positive CGC staining was found for all seven P2XRs and pannexins with IF microscopy and not visible after pre-incubation with competing peptides. mRNA expression was seen for each P2XR and pannexin as single bands at the expected base pair number. WB analysis revealed single bands for all P2XRs and pannexins at their correct molecular weight. [Ca²⁺]_i was significantly increased when stimulated with ATP. BzATP also significantly increased the [Ca²⁺]_i, although to a lesser value than ATP. Removing extracellular Mg²⁺ increased the ATP and BzATP response, but the P2X7 antagonist A804598 did not consistently alter these responses. Lowering the pH did not alter ATP or BzATP-induced [Ca²⁺]_i increase.

Conclusions : We conclude that all seven P2XRs and pannexins are present on cultured rat CGCs with P2X7, but not P2X2, appearing to be functional. The P2XRs offer new possible targets for regulating secretion of protective mucins.

This is a 2020 ARVO Annual Meeting abstract.