Abstract
Purpose :
Ionotropic purinergic receptors (P2XRs) are formed as hetero- or homotrimers from seven subunits, and are activated by extracellular nucleotides such as ATP. Upon activation, the P2XRs become permeable to cations, leading to activation of intracellular signaling pathways and an increase in cytosolic free calcium ([Ca2+]i). All P2XRs are present on cultured rat conjunctival goblet cells (CGCs). The purpose of this study was to investigate the source of the free cytosolic calcium in CGCs after the stimulation with ATP and the ATP analogues BzATP and 2MeSATP.
Methods :
Goblet cells were cultured from rat conjunctival explants. First passage CGCs were incubated with the ratiometric fluorescent dye Fura 2-AM in: 1. buffer containing calcium or in calcium free buffer with or without 2 mM EGTA, or 2. calcium-containing buffer with or without the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor thapsigargin (10-6 M) that depletes ER Ca2+ stores. The CGCs were then stimulated with the non-specific P2XR agonist ATP (10-7-10-5 M) or the ATP analogues BzATP (10-7-10-4 M) or 2MeSATP (10-7-10-5 M), and [Ca2+]i measured.
Results :
[Ca2+]i was significantly increased from baseline when stimulated with ATP (10-7-10-4 M), BzATP (10-7-10-4 M) or 2MeSATP (10-7-10-5 M), with a maximal response for ATP at 10-5 M, BzATP at 10-4 M and 2MeSATP at 10-5 M in buffer containing calcium. The change in peak [Ca2+]i was not significantly different in calcium free buffer, but was significantly reduced in the presence of 2 mM EGTA. The [Ca2+]i increase in CGCs preincubated with thapsigargin was completely reduced for ATP at 10-4 M, BzATP at 10-4 M and 2MeSATP at 10-5 M.
Conclusions :
We conclude that ATP and the ATP analogues BzATP and 2MeSATP increase [Ca2+]i mainly through releasing ER calcium stores.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.