Purinergic 2X 4 (P2X4), but not P2X7, receptors increase cytosolic [Ca < sup > 2+ < /sup > ] and stimulate mucin secretion in rat conjunctival goblet cells to maintain ocular surface health

Exp Eye Res. 2023 Aug 12:109614. doi: 10.1016/j.exer.2023.109614. Online ahead of print.ABSTRACTIonotropic purinergic receptors (P2XRs) are activated by ATP and ATP analogs. ATP can be released through ATP-permeable channels such as the pannexin hemichannels. Upon activation, the P2XRs become permeable to Ca2+, a potent stimulator of mucin secretion in conjunctival goblet cells (CGCs). The purpose of this study was to investigate the presence and function of P2XRs in CGCs. We also examined the presence of pannexin hemichannels. Rat first passage CGCs were stained with the goblet cell marker anti-cytokeratin 7 antibody and specific antibodies to P2X1-7 receptors and pannexin 1-3. mRNA expression was determined by RT-PCR using primers specific to P2XRs and pannexins. Proteins were identified with Western blotting (WB) using the same antibodies as for immunofluorescence (IF) microscopy. To study receptor function, CGCs were incubated with Fura 2-AM, exposed to agonists and antagonists, and intracellular [Ca2+] ([Ca2+]i) measured. [Ca2+]i was also measured after knock down of P2X4 and P2X7 receptor expression, and when exploiting P2XR specific characteristics. Lastly, mucin secretion was measured after the addition of several P2XR agonists. All P2XRs and pannexins were visualized with IF microscopy, and identified with RT-PCR and WB. [Ca2+]i was significantly increased when stimulated with ATP (10-7-10-4 M). Suramin, a non-selective P2XR antagonist at 10-4 M did not reduce ATP-in...

Source: Experimental Eye Research - August 14, 2023 Category: Opthalmology Authors: Haakon K Fj ærvoll Ketil A Fj ærvoll Menglu Yang Jeffrey Bair Tor P Utheim Darlene A Dartt Source Type: research