Objectives: Ameloblasts are the cells responsible for enamel formation (amelogenesis). Although the central role of ameloblasts in the synthesis and resorption of enamel matrix proteins has been well documented, Ca²⁺ transport mechanisms (via Ca²⁺ mobilization/extrusion) during amelogenesis have not been fully elucidated. In order to clarify the role of Ca²⁺ transporting/signaling mechanisms in rat ameloblasts, we investigated the presence and the functional characteristics of Na⁺/Ca²⁺ exchangers (NCX) which tightly regulate intracellular Ca²⁺ concentration ([Ca²⁺]_i). Methods: [Ca²⁺]_i was measured by fura-2 fluorescence in acutely isolated ameloblasts from newly born rats. The NCX activities were judged by their ability to carry out Ca²⁺ influx representing reverse Na⁺/Ca²⁺ exchange and measured with fura-2. In addition, total RNA was isolated from primary cultured ameloblasts, and RT-PCR analysis was performed to identify for the presence of transcripts for the NCX isoforms NCX1, NCX2 and NCX3. For immunohistochemical studies, cryostat sections of 6 µm thickness were prepared by slicing of the mandibles including incisors. These sections were incubated with monoclonal antibodies against NCX, and labeled using fluorescent secondary antibodies. Results: Ca²⁺ influx via reverse exchange was activated when Na⁺ was rapidly replaced with Li⁺ in the extracellular solution while maintaining osmotic strength. Ca²⁺ influx via reverse exchange showed a dependence on extracellular Ca²⁺ concentration. RT-PCR analysis revealed the expression of NCX1 and 3 mRNA in ameloblasts. Immunohistochemistry showed localization of NCX immunoreactivity on the ameloblast plasma membranes. Conclusions: Our results indicate significant expression of NCX in ameloblasts. Ca²⁺ extrusion via Na⁺/Ca²⁺ exchangers may serve as a directional Ca²⁺ transporting pathway to the enamel mineralizing front from circulation, and may also play an important role in the regulation of the intracellular Ca²⁺ signaling for both enamel matrix protein secretions and mineralization. (Supported by Grant-in-aid for research from the MEXT of Japan) Email: okumurar777@tdc.ac.jp

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