Using aldehyde-fixed rat incisor enamel organ, we localized Ca-ATPase activity ultracytochemically in the plasma membranes, the mitochondrial inner membranes, and the Golgi membranes of secretory ameloblasts and the cells of stratum intermedium at the secretory stage and papillary layer cells at the maturation stage, but not in maturation ameloblasts. This Ca-ATPase activity was totally dependent on substrate ATP, the enzyme activator CaCl2, and also sensitive to the specific calmodulin blocker trifluoperazine (TFP) in the incubation media. Specific antigenic sites of endogenous calmodulin were demonstrated in polyribosomes, the nucleus, mitochondria, and the cytoplasmic matrix along the plasma membranes of secretory ameloblasts, by the protein A-immunogold technique using sheep antiserum against bovine testis calmodulin. All other enamel organ cells-such as stratum intermedium, papillary layer cells, and maturation ameloblasts-were also weakly immunoreactive. In control sections incubated with antiserum pre-absorbed with an excess of calmodulin and protein A-gold complex, only a few gold particles were observed to be randomly associated with the tissues. Daily intraperitoneal injection of TFP (1 and 5 mg per 100 g body weight) for one week resulted in prominent migration of mitochondria from the infranuclear to supranuclear regions of secretory ameloblasts but caused no other morphological alterations in the enamel organ cells. EDX analysis of ultrathin sections revealed significantly lower peaks of Ca and P in the forming enamel of TFP-injected rats than those in controls. However, little reduction in the Ca and P levels in the maturing enamel was observed in TFP-injected rats. When growing enamel surfaces were exposed with NaOCl and examined with SEM, a remarkable defect in the enamel matrix was observed in the forming enamel but not in the maturing enamel. These results suggest that early enamel mineralization is dependent upon an intact calmodulin-regulated Ca-transporting ATPase in secretory ameloblasts and that enamel maturation is controlled by different mechanism(s).
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