Purinergic 2X 4 (P2X4), but not P2X7, receptors increase cytosolic [Ca2+] and stimulate mucin secretion in rat conjunctival goblet cells to maintain ocular surface health

Abstract

Ionotropic 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-induced peak [Ca²⁺]_i. The potent P2X7 agonist, BzATP (10^-7-10^-4 M) increased the [Ca²⁺]_i, although to a lesser extent than ATP. When measuring [Ca²⁺]_i the effect of repeated applications of ATP at 10^-5 or 10^-6 M the response "desensitized" after 30-60 s. The P2X4 specific antagonist 5-BDBD decreased the P2X4 agonist, 2MeSATP,-induced [Ca²⁺]_i increase. Furthermore, siRNA against the P2X4R, but not the P2X7R, decreased agonist-induced peak [Ca²⁺]_i. ATP (10^-5 M), BzATP (10^-4 M) and 2MeSATP (10^-5 M) induced mucin secretion. We conclude that all seven P2XRs are present in cultured rat CGCs. Of the P2XRs, only activation of the homotrimeric P2X4R appears to increase [Ca²⁺]_i and induce mucin secretion. The P2X4R in CGCs offers a new therapeutic target for protective mucin secretion.

Keywords: ATP; Calcium; Conjunctiva; Goblet cells; Inflammation; Ocular surface; Pannexins; Purinergic receptors.