Purinergic agonists increase [Ca] in rat conjunctival goblet cells through ryanodine receptor type 3.

ATP and benzoylbenzoyl-ATP (BzATP) increase free cytosolic Ca concentration ([Ca]) in conjunctival goblet cells (CGCs) resulting in mucin secretion. The purpose of this study was to investigate the source of the Ca mobilized by ATP and BzATP. First-passage cultured rat CGCs were incubated with Fura-2/AM, and [Ca] was measured under several conditions with ATP and BzATP stimulation. The following conditions were used: ) preincubation with the Ca chelator EGTA, ) preincubation with the SERCA inhibitor thapsigargin (10 M), which depletes ER Ca stores, ) preincubation with phospholipase C (PLC) or protein kinase A (PKA) inhibitor, or ) preincubation with the voltage-gated calcium channel antagonist nifedipine (10 M) and the ryanodine receptor (RyR) antagonist dantrolene (10 M). Immunofluorescence microscopy (IF) and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were used to investigate RyR presence in rat and human CGCs. ATP-stimulated peak [Ca] was significantly lower after chelating Ca with 2 mM EGTA in Ca-free buffer. The peak [Ca] increase in CGCs preincubated with thapsigargin, the PKA inhibitor H89, nifedipine, and dantrolene, but not the PLC inhibitor, was reduced for ATP at 10 M and BzATP at 10 M. Incubating CGCs with dantrolene alone decreased [Ca] and induced CGC cell death at a high concentration. RyR3 was detected in rat and human CGCs with IF and RT-qPCR. We conclude that ATP- and BzATP-induced Ca increases originate from the ER and that RyR3 may be an essential regulator of CGC [Ca]. This study contributes to the understanding of diseases arising from defective Ca signaling in nonexcitable cells. ATP and benzoylbenzoyl-ATP (BzATP) induce mucin secretion through an increase in free cytosolic calcium concentration ([Ca]) in conjunctival goblet cells (CGCs). The mechanisms through which ATP and BzATP increase [Ca] in CGCs are unclear. Ryanodine receptors (RyRs) are fundamental in [Ca] regulation in excitable cells. Herein, we find that ATP and BzATP increase [Ca] through the activation of protein kinase A, voltage-gated calcium channels, and RyRs, and that RyRs are crucial for nonexcitable CGCs' Ca homeostasis.