Dale R. Eisenmann

Cytochemical localization of Ca2+-Mg2+ adenosine triphosphatase in rat incisor ameloblasts during enamel secretion and maturation.

A modified Wachstein-Meisel medium containing lead or cerium as capturing ions was used to localize Ca2+-Mg2+ adenosine triphosphatase (ATPase; EC 3.6.1.3) in rat incisor ameloblasts during enamel formation. Sections representing different developmental stages were processed for electron microscopic cytochemistry. Distribution and intensity of the observed reaction product, which was almost exclusively associated with cell membranes, varied according to the stage of enamel formation. During the secretory stage, intense reaction product was evident along the entire plasma membrane of ameloblasts and papillary cells. The early transitional ameloblasts showed reaction product on their proximal and lateral cell membranes, but not distally. In late transitional (pre-absorptive) ameloblasts, distal cell membranes exhibited intense reaction product. During enamel maturation, smooth-ended ameloblasts showed reaction product proximally and laterally, but not distally. Ruffle-ended maturative ameloblasts exhibited intense reaction product along their lateral and distal membranes. The intensity of the latter was decreased but not eliminated by levamisole. In the transition from smooth-ended to ruffle-ended cells, the reaction product became evident distally, concomitant with the appearance of cell membrane invaginations. These data are consistent with a possible role for Ca2+-Mg2+ ATPase in controlling calcium availability at the enamel mineralization front.

Expression of plasma membrane Ca++ pump epitopes parallels the progression of enamel and dentin mineralization in rat incisor

We investigated the expression of Ca++ pump epitopes during enamel and dentin mineralization in the rat incisor. Secretory and maturation ameloblasts were studied as well as odontoblasts, using a monoclonal antibody (5F10) against human erythrocyte plasma membrane Ca++, Mg(++)-ATPase. A progressive increase in staining intensity in ameloblasts and the odontoblasts was observed beginning with the onset of mineralization. The mainly membrane-related labeling of ameloblasts showed variable intensity depending on the stage of enamel formation, whereas that of the odontoblasts showed even intensity during continued dentinogenesis. Staining of papillary cells was evident only during enamel maturation. Western blot analysis of freeze-dried ameloblasts was also used to determine the molecular weight of the Ca++ pump epitopes as well as the distribution and relative concentration of epitopes at each stage. An immunoreactive band of MW 140 KD and lower molecular weight bands that are more intense in late than in early maturation were demonstrated. Our studies suggest that the expression of plasma membrane Ca++ pump parallels the progression of mineralization in rat incisor enamel and dentin.

MORPHOMETRY AND AUTORADIOGRAPHY OF ALTERED RAT ENAMEL PROTEIN PROCESSING DUE TO CHRONIC EXPOSURE TO FLUORIDE

Female Sprague-Dawley rats had 6 weeks of 0 (control), 75 or 100 parts/10(6) sodium fluoride in their drinking water. Whole mandibular incisors were removed, fixed, demineralized and sections prepared for light-microscopic morphometric analysis of dose-related alterations in enamel protein retention. Other rats given 0 and 75 parts/10(6) only (control and experimental groups) were used for autoradiographic evaluation of alterations in enamel protein removal 35S-methionine was applied directly over secretory ameloblasts at the end of the fifth week of fluoride exposure. Incisors were removed either 5 or 7 days later and processed for autoradiographic analysis. The results indicated: (1) extended retention of enamel proteins in fluoride-exposed maturation enamel as well as reduced enamel protein synthesis and/or secretion in the secretory stage; (2) negative linear correlation between extended enamel protein retention and reduced enamel protein secretion among groups; and (3) repression of enamel protein removal. The data are also consistent with the concept that the fluoride effect is multifactorial.

Localization of Plasma Membrane Ca2+ Pump mRNA and Protein in Human Ameloblasts by In Situ Hybridization and Immunohistochemistry

The distribution of the plasma membrane Ca(2+)-pump (PMCA) proteins in human ameloblasts was examined immunohistochemically using monoclonal antibodies JA8 and 5F10. Further, the distribution of mRNA transcripts derived from two PMCA genes, PMCA-1 and PMCA-4 was examined using in situ hybridization. In rats, the PMCA-1 gene is purported to code for PMCA proteins with a role in maintaining the intracellular Ca2+ levels in nonepithelial cells. Other genes including the PMCA-4 gene may code for PMCA proteins characteristic of Ca2+ transporting epithelia. The present results show immunohistochemical staining in the Tomes processes and plasma membranes of human ameloblasts. Our studies also demonstrate a gradation of expression of the PMCA-1 and PMCA-4 mRNA transcripts which parallels the onset and progression of enamel mineralization. These studies suggest that PMCA proteins in human ameloblasts may function both in intracellular Ca2+ homeostasis and in regulating the vectorial Ca2+ influx into mineralizing enamel.

Calcium distribution in freeze-dried enamel organ tissue during normal and altered enamel mineralization

SummaryEnergy dispersive X-ray microanalysis was applied to freeze-dried blocks of enamel organ tissue to determine levels of calcium in various celular regions. The tissue blocks were dissected free from adjacent forming enamel following injection of cobalt or fluoride ions, both of which temporarily inhibit enamel mineralization. In all control and experimental specimens there was an increasing gradient of calcium from the stratum intermedium cells to the distal ends of the ameloblasts. Calcium levels were significantly reduced near the distal ends of the ameloblasts following cobalt or fluoride injection as compared with controls. It is suggested that evidence of an intercellular buildup of calcium near the distal ends of the ameloblast supports a controlling function of these cells. The changes in calcium levels are correlated with alterations in mineralization known to occur in the adjacent enamel of the model systems employed.

Quantitative cytochemistry of lysosomal structures in rat incisor maturation enamel organ

Trimetaphosphatase was used as a lysosomal marker in the ruffle-ended maturation ameloblasts and associated papillary cells. Morphometric analysis was carried out of the percentage area of these cells (density) occupied by the various enzyme-reactive lysosomal structures. The density of total TMPase-positive lysosomal structures, tubular lysosomes and multivesicular bodies in ruffle-ended ameloblasts were all significantly greater (p less than or equal to 0.05) in early than in late maturation enamel formation. In papillary cells the same was true of tubular lysosomes, whereas the greater density of enzyme-positive total structures in early maturation was not statistically significant when compared to late maturation. These findings demonstrate a corresponding pattern between enamel-organ lysosomal activity and the period of early enamel maturation when most enamel protein is lost. They support the likely involvement by ruffle-ended ameloblasts and papillary cells in absorption and degradation of exogenous enamel proteins.

A grinding method for producing sections of large areas of plastic embedded tissues

We have developed a method utilizing relatively thick ground sections of plastic embedded tissue which affords the resolution obtained with 0·5 μm cut sections. The sections, which are permanently affixed to plastic microscope slides, are much larger in area than ultramicrotome sections. Additional advantages are: sections can be destained and restained and selected areas can be examined with various forms of electron microscopy. Autoradiographic studies are also possible. Although the method has a broader application, it is particularly useful in examining the interface between hard and soft tissues.

Studies on the influx of [3H]-histidine and 45Ca through a surgical opening to rat incisor ameloblasts and adjacent enamel.

Considerable controversy exists about the role of ameloblasts in transport of calcium to mineralizing enamel. The rate and pattern of incorporation of calcium and an enamel precursor (histidine) were studied autoradiographically by introducing the isotopes through a surgically created defect in the lower border of the rat mandible. Influx of the isotopes to both secretory and maturation ameloblasts and adjacent enamel was examined in large survey sections of the entire incisor at intervals of 3 min to 2 h. Substantial concentrations of silver grains were observed over both secretory and maturation ameloblasts within 5 min of placement of either isotope. 45Ca was also present in secretory and maturation enamel within this short time. The overall patterns of influx and uptake of both precursors were similar to those found when such isotopes have been administered parenterally. The amount of influx of 45Ca across secretory, smooth-ended, and ruffle-ended maturation ameloblasts was compared qualitatively and found to be similar. The reproducibility of this surgical technique was demonstrated as well as its usefulness in combination with survey sections for multi-method investigations of rat incisor enamel formation and mineralization.

Quantitative Immunocytochemistry of Ca2+-Mg2+ ATPase in Ameloblasts Associated With Enamel Secretion and Maturation in the Rat Incisor

Our previous studies revealed intense membrane-associated labeling for Ca2+-Mg2+ ATPase (Ca2+-pump) in secretory and maturation ameloblasts in the rat incisor, both by enzyme cytochemistry and by immunohistochemical techniques. The purpose of the present study was to map the distribution of Ca2+-pump protein at the cellular and subcellular levels by means of a Ca2+-pump-specific monoclonal antibody and electron microscopic immunogold cytochemistry. Tissue specimens were dissected from secretory, early, and late enamel maturation zones. We quantified results by comparing gold particle densities over ameloblast lateral and distal plasma membrane regions, supranuclear cytoplasm, regions of the ruffled borders, and nuclei. The highest concentration of gold particles was seen over the distal membranes of early-maturation ameloblasts relative to those in late-maturation and secretory stages. Cytoplasmic labeling was less than that of the distal and lateral membranes, and gold particles located over nuclei were considered to be due to non-specific binding. These results are consistent with our earlier findings and suggest a role for the plasma membrane Ca2+-pump in the regulation of calcium availability to mineralizing enamel.

Cytochemical localization of calcium and Ca2+,Mg2(+)-adenosine triphosphatase in colchicine-altered rat incisor ameloblasts.

Colchicine is known to affect secretory, transport, and degradative functions of ameloblasts. The effects of colchicine on membrane-associated calcium and Ca2+,Mg2(+)-ATPase in secretory and maturation ameloblasts were investigated cytochemically. The pyroantimonate (PPA) method was used for localizing calcium and a modified Wachstein-Meisel medium was used to localize Ca2+,Mg2(+)-ATPase. Sections representing secretory and early maturation stages were examined by transmission electron microscopy. Morphological changes induced by colchicine included dislocated organelles and other well-established reactions to such anti-microtubule drugs. Calcium pyroantimonate (Ca-PA) deposits in most ameloblast types were markedly reduced, with the greater reduction occurring in those cells more severely altered morphologically. However, the cell membranes of both control and experimental smooth-ended maturation ameloblasts were essentially devoid of Ca-PA. The normal distribution and intensity of Ca2+,Mg2(+)-ATPase was not affected by colchicine. Because the observed reduction of membrane-associated calcium is apparently not mediated by Ca2+,Mg2(+)-ATPase in this case, other aspects of the calcium regulating system of ameloblasts are apparently targeted by colchicine.