The use of autoradiography and cycloheximide to determine the origin of enamel proteins in the maturation ameloblasts of the rat incisor.

Morphological and cytochemical studies have suggested that maturation ameloblasts participate in protein loss by absorbing and degrading enamel proteins as the enamel matures. Several immunocytochemical and autoradiographic studies have suggested other possible explanations for the presence of enamel matrix proteins in maturation ameloblasts. The weakness of these autoradiographic studies is the uncontrolled distribution of systemically injected radioactive amino acids, making it impossible to trace the source of the visualized intracellular isotope. This study used a localized technique to control the targets of the applied isotope and to identify enamel matrix proteins in the maturation ameloblasts with more confidence about their origin. The amount of labelled enamel protein was higher in maturation ameloblasts than transitional ameloblasts. When cycloheximide, a protein-synthesis inhibitor, was applied, there was no effect on the amount of labelled protein in the maturation ameloblasts. These findings support the hypothesis that maturation ameloblasts actively resorb and degrade enamel matrix proteins during enamel formation in the mandibular incisor of the rat.

Cyclic AMP-receptor proteins in the enamel matrix.

Cyclic AMP receptor proteins (cARP) are present in a variety of cell types. Intracellularly, they are the regulatory (R) subunits of type II cyclic AMP-dependent protein kinase (PKA: E.C.2.7.1.37). Additionally, cARP are secretory products of several cell types.[1] That cARP are present in and secreted by ameloblasts into the enamel matrix of the rat incisor was demonstrated by photoaffinity labeling, Western blotting and immunogold cytochemistry. Gold particles were present over cytoplasmic regions including Tomes' Processes of secretory ameloblasts, secretory granules and in the Golgi region. Specific RII labeling was seen in the enamel matrix, but not in dentin. The enamel matrix was more reactive during early maturation compared to the secretory stage of amelogenesis. Nuclear labeling with the RII antibody showed higher intensity in maturation than in secretory ameloblasts. These results demonstrate that cARP are expressed in ameloblasts and secreted into the enamel matrix. The role(s) of cARP in enamel matrix mineralization and the involvement of PKA-regulated pathways in enamel protein synthesis and secretion remain to be determined.

Quantitative immunocytochemistry of Ca(2+)-Mg2+ ATPase in ameloblasts associated with enamel secretion and maturation in the rat incisor.

Our previous studies revealed intense membrane-associated labeling for Ca(2+)-Mg2+ ATPase (Ca(2+)-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 Ca(2+)-pump protein at the cellular and subcellular levels by means of a Ca(2+)-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 Ca(2+)-pump in the regulation of calcium availability to mineralizing enamel.

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.

In situ hybridization and monoclonal antibody analysis of plasma membrane Ca-pump mRNA and protein in submandibular glands of rabbit, rat and man.

The degree of supersaturation of saliva with calcium (Ca) is related to the mineral phase of enamel in erupted teeth, the incidence of caries, and the formation of calculus. The mechanisms for regulating salivary Ca concentration are therefore of relevance to dentistry. Sections of rabbit, rat and human submandibular gland (SMG) were processed for immuno-histochemistry with a specific anti-plasma membrane Ca-pump antibody, 5F10. Western blots confirm that the molecular weight of the proteins identified by our antibody (135 kDa) is consistent with an appropriate molecular weight for PMCA antigen (135-150 kDa). Tissue sections were also processed for in situ hybridization to study the distribution of the PMCA mRNA isoforms. In mammals, the PMCA1 gene is reported to code for a PMCA protein with a role in maintaining the intracellular Ca levels in both epithelial and non-epithelial cells. Other genes including the PMCA2 and PMCA4 genes may code for PMCA proteins specific to Ca transporting tissues. Our studies demonstrate cytoplasmic labeling of PMCA mRNA with hPMCA-1 and hPMCA-4 specific cDNA probes in humans, and rPMCA-1 and rPMCA-2 specific oligonucleotide probes in rats. Labeling of PMCA protein and all mRNA isoforms was found in the cytoplasm of the interlobular and intralobular ducts (except for intercalated ducts). The demonstrated presence of PMCA in SMGs of rabbit, rat, and man, may suggest a role for PMCA in the regulation of intracellular Ca and in a mechanism for regulating and maintaining the high concentration of Ca in salvia.

Ameloblast cycling patterns as measured by fluorochrome infiltration of rat incisor enamel.

Rapid modulation of maturation ameloblasts between smooth-ended and ruffle-ended forms may play an important part in the development of normal dental enamel. Previous studies of modulation rates relied upon measurements of stained or fluorescing bands on the enamel surface of whole incisors along with separate histological sections for cell-band dimensions. The present study utilized direct measurement of maturation-ameloblast bands and fluorescing regions of underlying enamel in the same histological sections, which increased the accuracy and ease with which modulation rates could be determined. Rats were injected with calcein at various times before killing and preparation of survey midsagittal sections of the lower incisors. The lengths of bands of smooth-ended ameloblasts and underlying fluorescing regions of enamel were measured throughout the maturation zone. Modulation rates were found to range from 238 microns/h (early maturation) to 91 microns/h (late maturation). Calcein diffused into enamel to varying degrees depending upon the location within the maturation stage. This new approach of direct measurement greatly facilitates the investigation of ameloblast modulation and provides additional insights into progressive structural changes in enamel during maturation.

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.

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.

Effects of vinblastine on calcium distribution pattern and Ca2+,Mg(2+)-adenosine triphosphatase in rat incisor maturation ameloblasts.

Vinblastine is known to affect secretory and transport functions of ameloblasts. The effects of vinblastine on distribution patterns of membrane-associated calcium and Ca2+,Mg(2+)-ATPase in maturation ameloblasts were investigated cytochemically. The potassium pyroantimonate (PPA) method was used for localizing calcium and a modified Wachstein-Meisel medium was used to localize Ca2+,Mg(2+)-ATPase. Ultrastructural changes induced by vinblastine included dislocated organelles and reduction or elimination of the ruffled border of the ameloblasts. Membrane-associated calcium pyroantimonate deposits were markedly reduced. The intensity of Ca2+,Mg(2+)-ATPase reaction product was also markedly reduced by vinblastine. Concomitant reduction of membrane-associated calcium and Ca2+,Mg(2+)-ATPase lends support to a role for maturation ameloblasts in control of a cyclic pattern of influx of calcium to mineralizing enamel.

Fine structural changes and lysosomal phosphatase cytochemistry of ameloblasts associated with the transitional stage of enamel formation in the rat incisor.

Trimetaphosphatase (TMPase) and cytidine-5'-monophosphatase (CMPase) were used as lysosomal markers in the transitional ameloblasts (TA) to investigate the distribution of lysosomal structures and to correlate the cytochemical findings with the ultrastructural features of these cells. Of particular interest were the cytochemical and morphological changes which occur as the ameloblasts approach the maturation stage of enamel formation. The sequence of changes observed provides a basis for designation of three regions of the transitional zone (early and late TA and modulating ameloblasts). In the early TA region, the cells decreased in height and contained phagic vacuoles as well as numerous TMPase and CMPase reactive structures. Late transitional ameloblasts had invaginations at their distal ends as well as membrane-bound structures, both filled with fine granular material. Dense bodies, phagic vacuoles, and other elements of the lysosomal system were enzyme reactive. Modulating ameloblasts lacked the phagic vacuoles but exhibited large numbers of multivesicular bodies, vesicles, and secretory granules. Their distal ends were morphologically altered indicating a change towards ruffle- or smooth-ended varieties of maturation ameloblast. In the former, increased granular material was observed within cell membrane invaginations and associated membrane-bound structures. In the latter, intercellular spaces widened and were filled with granular material. The present cytochemical findings of an extensive lyosomal system in transitional ameloblasts confirm the function of those cells in reducing the secretory ameloblast population and in the selective elimination of their protein-synthesizing organelles. Furthermore, this extensive lysosmal system and the present morphological findings are consistent with a potential role for transitional ameloblasts in contributing to the marked loss of enamel protein known to occur during maturation.

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.

Effect of colchicine on lysosomal structures in maturation-ameloblasts of the rat incisor.

The lysosomal systems in maturation-ameloblasts affected by colchicine were examined using trimetaphosphatase cytochemistry. Demineralized segments of rat incisor were incubated for trimetaphosphatase. At all time intervals, lysosomal structures exhibited reduced enzyme reactivity and were clustered in the Golgi region of the cell. Both ruffle-ended and smooth-ended ameloblasts maintained essentially normal morphology up to 4 h after colchicine injection, except for some migration of organelles. After 8 h, the ruffled border was markedly modified and the associated dense granular material was no longer present. Changes in the lysosomal system and ruffled border indicate interference by colchicine with a putative resorptive function of the maturation-ameloblasts.

Tubular lysosomes in papillary cells during maturation of enamel in the rat.

TMPase and CMPase were used as lysosomal markers. Demineralized-incisor specimens were incubated for TMPase in a lead-acetate based medium and for CMPase in a modified medium where cerium was used as the capture ion. Papillary cells contained an extensive network of branched and interconnected tubular structures reactive for both TMPase and CMPase; there were some of these structures also in the endothelium of adjacent capillaries. Fine granular material was observed in these tubular structures, as well as in coated vesicles and multivesicular bodies in the papillary cells. These cytochemical results demonstrate the presence of tubular lysosomes in papillary cells, as have already been found in adjacent maturation ameloblasts. These structures may be elements of an extensive endosomal system involved in the degradation of enamel protein. These findings also support the concept that the various cells of the enamel organ constitute a functional unit.

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.

Effect of cobalt on Ca2+-Mg2+ ATPase in rat incisor maturation ameloblasts.

Rat incisor maturation ameloblasts were studied to determine the effect of injected cobalt on the distribution and intensity of Ca2+-Mg2+ ATPase. The dosage of cobalt utilized temporarily inhibits enamel mineralization and alters ameloblast-associated calcium. A modified Wachstein-Meisel medium containing cerium as the capturing ion was used to localize Ca2+-Mg2+ ATPase cytochemically. The distribution and intensity of the reaction product in normal maturation ameloblasts was, as previously reported, primarily in association with the plasma membranes. The lateral cell membranes of both smooth-ended and ruffle-ended ameloblasts were reactive. The ruffled border region contained the heaviest concentration of reaction product. Although cobalt did not alter the general pattern of distribution of the reaction product in either cell type, in all regions of activity the intensity was noticeably increased. Cells modulating from smooth-ended to ruffle-ended ameloblasts and under the influence of cobalt exhibited an irregular dense layer along the enamel surface, and large focal accumulations of electron-dense material in the various extracellular compartments. This may indicate interference with a putative resorptive activity of these cells.

Tubular lysosomes in ruffle-ended ameloblasts associated with enamel maturation in rat incisor.

Trimetaphosphatase (TMPase) and cytidine-5'-monophosphatase (CMPase) were localized to investigate the lysosomal system, particularly tubular lysosomes, in ruffle-ended ameloblasts associated with maturation of enamel in rat incisor. Demineralized specimens were incubated for TMPase and for CMPase in a modified medium where cerium was used as the capture ion. Ruffle-ended ameloblasts showed distal invaginations and membrane-bound bodies filled with fine granular material, some of which displayed CMPase reaction product. Elongated tubular configurations 80-140 nm wide were distributed throughout the cytoplasm and were reactive with both TMPase and CMPase, thus characterizing these structures as lysosomes. They often contained fine granular material morphologically similar to that present in multivesicular bodies. During late enamel maturation, fewer tubular lysosomes were observed when compared to early maturation. These cytochemical results demonstrate the presence of tubular lysosomes in ruffle-ended ameloblasts, and it is suggested that they are elements of the endosomal system in these cells. These findings are also consistent with a resorptive function for ruffle-ended ameloblasts during enamel maturation.

Calcium levels in ruffle-ended and smooth-ended maturation ameloblasts.

Scanning electron microscopy was used to distinguish the topographical characteristics of two maturation ameloblast types in freeze-dried blocks of enamel organ tissue. This distinction was based primarily upon the configuration of the distal ends of the ameloblasts and the presence or absence of wide intercellular spaces. Energy dispersive x-ray spectrometry was applied to compare calcium levels in various regions of tissue identified as constituting either ruffle-ended or smooth ended ameloblasts. Greater levels of calcium were found in the distal ends of the ruffle-ended cells than in their proximal ends. In addition, greater calcium levels were found in the distal ends of the ruffle-ended cells than the distal ends of the smooth-ended cells. The higher calcium levels in ruffle-ended cells correlates with the view that these cells are actively involved in control of movement of calcium to the enamel front.

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.

Effect of fluoride and cobalt on forming enamel: scanning electron microscope and X-ray microanalysis study.

The forming surfaces of enamel of rat incisors were examined by scanning electron microscope one hour after injection of either 5 mg/100 g body weight of sodium fluoride or 12 mg/100 g body weight of cobalt chloride. The cell debris from the surfaces of the separated incisors was either gently wiped off with soft facial tissues or chemically removed by treating with NaOH, NaOCl or trypsin. Best results to remove cell debris were obtained from 0.25% trypsin treatment. SEM studies revealed that the surface of the normal secretory enamel was characteristic in appearance with well-developed smooth prism outlines. In fluoride specimens the prism outlines were feathery in appearance, laced with protruding spine-shaped clusters of mineral crystals. In the case of cobalt treatment, prism outlines were less uniform and in some areas they were incomplete. The calcium concentration of surface enamel was significantly lower in the cobalt-treated specimens than those from control and fluoride-treated animals. The Ca:Mg ratio was also lower in cobalt-treated specimens as compared to control and fluoride-treated ones.

Secretory ameloblasts and calcium distribution during normal and experimentally altered mineralization.

The distribution of calcium in relation to secretory ameloblasts of the rat incisor was studied. An experimental model system in which enamel mineralization was temporarily inhibited by injecting sodium fluoride and cobalt chloride was used. Potassium pyroantimonate (PPA) cytochemistry, electron energy loss spectroscopy (EELS), and energy dispersive X-ray spectrometry (EDS) were used to clarify the role of the ameloblast in controlling calcium distribution during normal and experimentally altered enamel mineralization. Secretory ameloblasts chemically-preserved in glutaraldehyde either with or without PPA were analyzed for calcium; those preserved with PPA showed higher concentrations of calcium than did those preserved with glutaraldehyde only. Freeze-dried control and experimental tissues showed an increasing gradient of calcium from stratum intermedium cells to the distal ends of the ameloblasts. Calcium levels were reduced near the distal ends of the cells following fluoride and cobalt injections, while magnesium levels were increased markedly in the same region. This multi-method approach showed correlated calcium localization in specific regions of this cell in relation to changes in function. The study thus provides additional evidence for active involvement of the ameloblasts in enamel mineralization.