Extracellular ATP Mediates The Intercellular Ca2+ Wave Induced By Mechanical Stimulation In Human Salivary Gland Cells

Abstract

Intracellular Ca2+ signaling has a central role in regulation of salivary gland cell function. Coordination of Ca2+ signaling between cells contributes to synchronized and effective secretion of saliva. However, mechanisms that underlie this signaling remain elusive. Here, intercellular Ca2+ waves (ICW) and their propagation in human salivary gland (HSG) cells were investigated using fura-2 fluorescence imaging. While not well understood, mechanical stimulation of a single cell in a cluster with a micropipette induces ICW. The Ca2+ signal is propagated from the stimulated cell to the 7-9th tier of cells or ∼120 μm. The following findings indicate that ICW propagation in HSG cells uses an extracellular and ATP-dependent pathway. The purinergic receptor antagonist suramin significantly decreased ICW propagation. Extracellular ATP or UTP abolished ICW suggestive of receptor desensitization. Gap junction intercellular communication is not involved in ICW in HSG cells because the gap junction inhibitor oleamide did not inhibit ICW. Furthermore, HSG cells showed poor dye coupling upon microinjection of Lucifer Yellow. The Ca2+ transients observed within each cell are dependent on Ca2+ release from the ER as thapsigargin abolished the ICW. The phospholipase C inhibitor U73122 also blocks ICW indicating that these transients are IP3-dependent. Furthermore, store operated Ca2+ entry (SOCE) modulates the amplitude of Ca2+ signal since removal of extracellular Ca2+ or a SOCE inhibitor SK&F 96365 decreased the amplitude of Ca2+ signal. Inhibition of mitochondrial Ca2+ uptake with FCCP/oligomycin or ruthenium red showed similar effects on the amplitude. These results indicate that propagation of this ICW utilizes extracellular ATP, likely through the P2U(P2Y2) receptor in HSG cells. The major Ca2+ mobilization mechanisms are IP3-dependent ER Ca2+ release and SOCE. Finally, mitochondrial energy metabolism and Ca2+ uptake modulated this ICW propagation.