Mitochondria modulate ameloblast Ca²⁺ signaling

The role of mitochondria in enamel, the most mineralized tissue in the body, is poorly defined. Enamel is formed by ameloblast cells in two main sequential stages known as secretory and maturation. Defining the physiological features of each stage is essential to understand mineralization. Here, we analyzed functional features of mitochondria in rat primary secretory and maturation-stage ameloblasts focusing on their role in Ca²⁺ signaling. Quantification of the Ca²⁺ stored in the mitochondria by trifluoromethoxy carbonylcyanide phenylhydrazone stimulation was comparable in both stages. The release of endoplasmic reticulum Ca²⁺ pools by adenosine triphosphate in rhod2AM-loaded cells showed similar mitochondrial Ca²⁺ (_mCa²⁺) uptake. However, _mCa²⁺ extrusion via Na⁺-Li⁺-Ca²⁺ exchanger was more prominent in maturation. To address if _mCa²⁺ uptake via the mitochondrial Ca²⁺ uniporter (MCU) played a role in cytosolic Ca²⁺ (_cCa²⁺) buffering, we stimulated Ca²⁺ influx via the store-operated Ca²⁺ entry (SOCE) and blocked MCU with the inhibitor Ru265. This inhibitor was first tested using the enamel cell line LS8 cells. Ru265 prevented _cCa²⁺ clearance in permeabilized LS8 cells like ruthenium red, and it did not affect ΔΨm in intact cells. In primary ameloblasts, SOCE stimulation elicited a significantly higher _mCa²⁺ uptake in maturation ameloblasts. The uptake of Ca²⁺ into the mitochondria was dramatically decreased in the presence of Ru265. Combined, these results suggest an increased mitochondrial Ca²⁺ handling in maturation but only upon stimulation of Ca²⁺ influx via SOCE. These functional studies provide insights not only on the role of mitochondria in ameloblast Ca²⁺ physiology, but also advance the concept that SOCE and _mCa²⁺ uptake are complementary processes in biological mineralization.