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

  • Invest Ophthalmol Vis Sci. 2025 Jan 2;66(1):46. doi: 10.1167/iovs.66.1.46.
Ketil A Fjærvoll  1  2  3  4 ,  Haakon K Fjærvoll  1  2  3  4 ,  Menglu Yang  1 ,  Jeffrey Bair  1 ,  Tor P Utheim  1  2  3  4 ,  Darlene A Dartt  1
Affiliations
  • 1. Schepens Eye Research Institute, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, United States.
  • 2. Division of Head, Neck and Reconstructive Surgery, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway.
  • 3. Medical Student Research Program, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway.
  • 4. Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway.
PMID: 39836405 DOI: 10.1167/iovs.66.1.46
Abstract

Purpose: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

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