Ca²⁺ Extrusion via Na⁺/Ca²⁺ Exchanger Subtype 3 in Rat Odontoblasts

Objectives: Ca²⁺ is essential for the signal transduction pathway, and odontoblasts express several Ca²⁺ mobilization mechanisms such as; 1) IP₃-induced Ca²⁺-release by PLC-coupled receptor activations, 2) depolarization-mediated Ca²⁺ mobilization and 3) Ca²⁺ influx via store-operated Ca²⁺ channels. [Ca²⁺]_i levels are tightly regulated by Ca²⁺ extrusion system including Na⁺/Ca²⁺ exchangers (NCX) in the plasma membrane. In this study, we investigated the functional characteristics of the NCX in rat odontoblasts. Methods: We have characterized both reverse (Ca²⁺ influx) and forward (Ca²⁺ extrusion) modes of NCX activities in odontoblasts on a dental pulp slice preparation. The reverse NCX activity was judged by their ability to carry out Ca²⁺ influx, and [Ca²⁺]i was measured by fura-2 fluorescence. The forward one carried inward current with Na⁺, and was measured by a nystatin perforated-patch clamp recording. For immunohistochemical studies, cryostat sections (6 ìm thickness) prepared from the mandibles including incisors were incubated with monoclonal antibodies against NCX, and labeled using fluorescent secondary antibodies. Results: Ca²⁺ influx by reverse exchange showed a dependence on extracellular Ca²⁺ (K_D = 0.23 mM). NCX inhibitor, KB-R7943, for all NCX isoforms (NCX1, NCX2 and NCX3) blocked reverse exchange in a concentration-dependent manner (IC₅₀ = 2.4 μM). However NCX1- and NCX2-specific inhibitor, SEA0400, had no effects on the reverse exchange, showing that odontoblasts express NCX3. Inward currents via forward exchange showed dependence on external Na⁺, with a K_D of 44 mM. Immunohistochemical localization of NCX was detected at distal membrane of odontoblasts. Conclusion: Our results indicate significant expression of NCX3 in odontoblasts. Coupling mechanism between Na⁺-Ca²⁺ exchange at distal membrane facing dentin mineralizing front may serve as a directional Ca²⁺ transport pathway from the circulation, after intracellular Ca²⁺ signaling events. This work was supported by HRC7, HRC6A03 projects, and Grant-in-Aid (No. 18592050) for Scientific Research from the MEXT of Japan.