Identification of Purinergic Type 2 Y Receptors (P2YRs) in Conjunctival Goblet Cells

Abstract

Purpose : P2Y receptors (P2YRs) are G protein-coupled receptors activated by nucleotides and are of significance in fluid and mucous secretion in the conjunctiva. P2Y2 receptor agonists have been developed for treatment of dry eye. The purpose of this study is to elucidate which P2YRs are present in rat conjunctival goblet cells (CGCs) and determine the effects of their activation on the intracellular Ca²⁺ concentration ([Ca²⁺]_i).

Methods : Conjunctiva from male Sprague-Dawley rats was minced and placed in six-well plates for culture of CGCs. To investigate expression of P2YRs mRNA was extracted from CGCs and used for RT-PCR with primers specific for all P2YRs present in the rat genome. Immunofluorescence experiments were performed using first passage CGCs seeded overnight on glass coverslips, fixed with paraformaldehyde and stained with antibodies specific to P2YRs. The cells were co-labeled with the CGC marker anti-cytokeratin 7 (CK7) antibody. Western blot analyses were done using proteins extracted from first passage CGCs and receptor-specific antibodies. To indicate function, first passage CGCs were incubated with the Ca²⁺ specific ratiometric dye Fura2-AM and [Ca²⁺]_i measured after cells were stimulated with the P2YR selective agonists UTP and ADP or agonists specific to P2Y1R (MRS2365), P2Y2R (MRS2768), P2Y4R (MRS4062) or P2Y6R (MRS2693). Calcium experiments were done in triplicate and all experiments were performed on CGCs from at least three rats.

Results : mRNA for all P2Y receptors was expressed in cultured goblet cells as shown by single bands at the correct base pair numbers. Protein for the same receptors were each present as a single band at the correct molecular weight, except for the P2Y2R which had 2 bands. All P2YRs were visualized in the CGCs. Pre-incubation with competing peptides was followed by absence of all P2YRs except P2Y2R. The P2Y2, 4 and 6 agonist UTP and the P2Y1, 12 and 13 agonist ADP increased the [Ca²⁺]_i in a concentration-dependent manner. UTP was the most potent. Receptor-specific agonists increased [Ca²⁺]_i, although to a level substantially lower than the UTP response at 10^-3 M.

Conclusions : We conclude that P2Y agonists are potent stimuli of CGC function and likely work through multiple receptors as all the P2YRs are present in these cells.

This is a 2020 ARVO Annual Meeting abstract.