Purinergic agonists increase [Ca2+]i in rat conjunctival goblet cells through ryanodine receptor type 3.

Abstract

ATP and benzoylbenzoyl-ATP (BzATP) increase free cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in conjunctival goblet cells (CGCs) resulting in mucin secretion. The purpose of this study was to investigate the source of the Ca²⁺_i mobilized by ATP and BzATP. First-passage cultured rat CGCs were incubated with Fura-2/AM, and [Ca²⁺]_i was measured under several conditions with ATP and BzATP stimulation. The following conditions were used: 1) preincubation with the Ca²⁺ chelator EGTA, 2) preincubation with the SERCA inhibitor thapsigargin (10^-6 M), which depletes ER Ca²⁺ stores, 3) preincubation with phospholipase C (PLC) or protein kinase A (PKA) inhibitor, or 4) preincubation with the voltage-gated calcium channel antagonist nifedipine (10^-5 M) and the ryanodine receptor (RyR) antagonist dantrolene (10^-5 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 [Ca²⁺]_i was significantly lower after chelating Ca²⁺_i with 2 mM EGTA in Ca²⁺-free buffer. The peak [Ca²⁺]_i increase in CGCs preincubated with thapsigargin, the PKA inhibitor H89, nifedipine, and dantrolene, but not the PLC inhibitor, was reduced for ATP at 10^-5 M and BzATP at 10^-4 M. Incubating CGCs with dantrolene alone decreased [Ca²⁺]_i 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 Ca²⁺_i increases originate from the ER and that RyR3 may be an essential regulator of CGC [Ca²⁺]_i. This study contributes to the understanding of diseases arising from defective Ca²⁺ signaling in nonexcitable cells.NEW & NOTEWORTHY ATP and benzoylbenzoyl-ATP (BzATP) induce mucin secretion through an increase in free cytosolic calcium concentration ([Ca²⁺]_i) in conjunctival goblet cells (CGCs). The mechanisms through which ATP and BzATP increase [Ca²⁺]_i in CGCs are unclear. Ryanodine receptors (RyRs) are fundamental in [Ca²⁺]_i regulation in excitable cells. Herein, we find that ATP and BzATP increase [Ca²⁺]_i through the activation of protein kinase A, voltage-gated calcium channels, and RyRs, and that RyRs are crucial for nonexcitable CGCs' Ca²⁺_i homeostasis.