Pyrimidinergic P2Y1-Like Nucleotide Receptors Are Functional in Rat Conjunctival Goblet Cells

To investigate the presence of uridine-5'-triphosphate (UTP)-activated P2Y1-like nucleotide receptors (P2Y2R, P2Y4R, and P2Y6R) in conjunctival goblet cells (CGCs) and determine if they increase intracellular Ca2+ concentration ([Ca2+]i) and induce mucin secretion.

Adult, male rat conjunctiva was used for culture of CGCs. To investigate the expression of P2YRs, mRNA was extracted from CGCs and used for reverse transcription PCR (RT-PCR) with commercially obtained primers specific to P2Y2R, P2Y4R, and P2Y6R. Immunofluorescence (IF) and western blot (WB) analyses were performed using first-passage CGCs and stained with antibodies specific to each P2YR. Furthermore, CGCs were incubated with fura-2/AM, and [Ca2+]i was measured after stimulation with the P2YR selective agonists UTP, uridine 5'-diphosphate (UDP), or UDP-glucose and agonists specific to P2Y2R (MRS 2768), P2Y4R (MRS 4062), and P2Y6R (MRS 2693). [Ca2+]i measurements after P2Y2R and P2Y6R siRNA treatment were performed. Mucin secretion was measured after stimulation of P2Y2R, P2Y4R, and P2Y6R.

mRNA for all pyrimidinergic P2Y1-like receptors was found as single bands of expected base pair number with RT-PCR. The presence of these P2YRs was confirmed with IF microscopy and WB analysis. UTP and UDP elicited concentration-dependent increases in [Ca2+]i. The receptor-specific agonists and UDP-glucose increased [Ca2+]i, although these responses were substantially lower than those elicited by UTP and UDP at 10-4 M and 10-3 M and did not show similar dose dependency. P2Y2R- and P2Y6R-depleted CGCs responded with reduced peak [Ca2+]i. UTP, MRS 2768 (P2Y2R), and UDP each stimulated mucin secretion from CGCs.

P2Y2R, P2Y4R, and P2Y6R are present and functional in rat CGCs and may represent novel therapeutic targets for dry eye treatment and other types of ocular surface disease.