Histochemical demonstration and microanalysis of possible calcium binding sites in the enamel organ of rat incisors.

The rat incisors obtained from rats perfused with high-calcium solution containing 30 to 50 mM CaCl2 were processed for rapid freeze/freeze-substitution and embedded in epoxy resin. GBHA staining, a histochemical staining for calcium, or unhydrously prepared sections revealed a large number of granular Ca-GBHA reactions in the enamel organ, most of which being located along the lateral plasma membranes of the ameloblasts. In the ameloblast layer, the reaction was negative in the presecretory stage, became intense in concert with the onset of enamel matrix formation, and remained so by the end of the transitional stage where the reaction gradually diminished. In the maturation stage, similar GBHA reactions emerged only in association with the smooth-ended ameloblasts and were absent in the ruffle-ended ameloblasts. Some granular reactions of much smaller diameter were occasionally noted in other cells of the enamel organ at both the secretory and maturation stages. Electron microscopy confirmed large electron-dense granules, each showing distinct peaks for Ca and P by microanalysis, associated with the cytoplasmic aspect of lateral plasma membranes of secretory, transitional, and smooth-ended ameloblasts. The present study thus revealed a strong calcium-binding property of the cytoplasmic aspect of lateral plasma membranes of secretory ameloblasts and its fluctuation in the maturation ameloblasts under the given experimental conditions. A necessity for an extensive analysis of membrane constituents of the enamel organ was proposed for better understanding of cellular roles in calcium regulation during amelogenesis.