Differential Expression of the Tight Junction Proteins,Claudin‐1, Claudin‐4, Occludin,ZO‐1, and PAR3, in the Ameloblasts of Rat Upper Incisors

Tight junctions (TJs) create a paracellular permeability barrier to restrict the passage of ions, small solutes, and water. Ameloblasts are enamel‐forming cells that sequentially differentiate into preameloblasts, secretory, transition, and ruffle‐ended and smooth‐ended maturation ameloblasts (RAs and SAs). TJs are located at the proximal and distal ends of ameloblasts. TJs at the distal ends of secretory ameloblasts and RAs are well‐developed zonula occludens, but other TJs are moderately developed but incomplete zonula occludens (ZO) or less‐developed macula occludens. We herein examined the immunofluorescence localization of TJ proteins, 10 claudin isoforms, occludin, ZO‐1, and PAR3, a cell polarity‐related protein, in ameloblasts of rat upper incisors. ZO‐1 and claudin‐1 were detected at both ends of all ameloblasts except for the distal ends of SAs. Claudin‐4 and occludin were detected at both ends of transition and maturation ameloblasts except for the distal ends of SAs. PAR3 was detected at the proximal TJs of all ameloblasts and faintly at the distal TJs of early RAs. These results indicate that functional zonula occludens formed at the distal ends of the secretory ameloblasts and RAs consisted of different TJ proteins. Therefore, the distal TJs of secretory ameloblasts and RAs may differentially regulate the paracellular permeability to create a microenvironment suitable for enamel deposition and enamel maturation, respectively. In addition, PAR3 may be principally involved in the formation and maintenance of the proximal, but not distal, TJs. Anat Rec, 291:577–585, 2008. © 2008 Wiley‐Liss, Inc.     

Immunocytochemical localization of amelogenins in the deciduous tooth germs of the human fetus

The immunocytochemical localization of amelogenins in the developing deciduous tooth germs of 6-month-old human fetuses was investigated by the protein A-gold method using an antiserum against porcine 25K amelogenin. The inner enamel epithelial cells and underlying matrix showed no amelogenin-like immunoreactivity. Distinct immunoreactivity was initially shown by fine fibrils found beneath the intact basal lamina of preameloblasts at the early differentiation stage. At the late differentiation stage, amelogenin-like immunoreactivity was shown by a fine granular material within the extracellular matrix as well as by the Golgi apparatus, secretory granules, lysosomal structures, coated vesicles, and coated pits of preameloblasts with a disrupted basal lamina. At the formative stage, the localization of immunoreactivity in secretory ameloblasts was similar to that in preameloblasts during the late differentiation stage. However, immunopositive coated vesicles and coated pits were only found at the early stage of matrix formation. The calcified enamel matrix and stippled material showed intense immunoreactivity. Immunocytochemical labeling of the enamel matrix appeared as a gradient, decreasing from the enamel surface to the dentinoenamel junction. No maturation stage of ameloblasts existed in the tooth germs examined. In predentin and dentin, amelogenin-like immunoreactivity was occasionally detected on odontoblasts and their processes, but odontoblasts and cells of the stratum intermedium contained no immunoreactive elements. These findings confirmed that the secretory ameloblast in the human deciduous tooth germ is responsible for the synthesis and secretion of enamel proteins.     

Cyclic AMP-Receptor Proteins in the Enamel Matrix

Cyclic AMP receptor proteins (cARP) are present in a variety of cell types. Intra-cellularly, 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.     

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.     

Differential expression patterns of the tight junction-associated proteins occludin and claudins in secretory and mature ameloblasts in mouse incisor

Tight junctions (TJs) function primarily as a barrier against paracellular transport between epithelial cells and are composed mainly of occludin (OLD) and claudins (CLDs). The CLD family consists of 24 members that show tissue- or cell-specific expression. Ameloblasts, which originate from the oral epithelium, form enamel, and enamel proteins and minerals are transported across the ameloblastic layer during amelogenesis. We immunohistochemically examined the distribution patterns of TJs in ameloblasts by observing the expression patterns of OLD and CLDs (CLD-1 to CLD-10). Secretory ameloblasts contained OLD and CLD-1, -8, and -9 at the distal end of the cell. In mature ameloblasts, OLD and CLD-1, -6, -7, -8, -9, and -10 were present mainly at both the distal and proximal ends of the cell, regardless of whether the ameloblasts were ruffle-ended or smooth-ended. Mature ameloblasts in which only the proximal ends were stained for OLD and CLDs were also found. These results indicate that the expression patterns of CLDs and the distribution patterns of TJs change drastically between the secretory and mature ameloblast stages, suggesting that these patterns reflect the different functions of these cells, specifically in the transport of proteins and ions for enamel formation.     

The dentino-enamel junction revisited

The dentino-enamel junction is not an simple inert interface between two mineralized structures. A less simplistic view suggests that the dentino-enamel junctional complex should also include the inner aprismatic enamel and the mantle dentin. At early stages of enamel formation, fibroblast growth factor (FGF)-2 is stored in and released from the inner aprismatic enamel, possibly under the control of matrix metalloproteinase (MMP)-3. The concentration peak for MMP-2 and -9 observed in the mantle dentin coincided with a very low labeling for TIMP-1 and -2, favoring the cross-talk between mineralizing epithelial and connective structures, and as a consequence the translocation of enamel proteins toward odontoblasts and pulp cells, and vice versa, the translocation of dentin proteins toward secretory ameloblasts and cells of the enamel organ. Finally, in X-linked hypophosphatemic rickets, large interglobular spaces in the circumpulpal dentin were the major defect induced by the gene alteration, whereas the mantle dentin was constantly unaffected. Altogether, these data plead for the recognition of the dentino-enamel junctional complex as a specific entity bearing its own biological characteristics.     

The Dentino-Enamel Junction Revisited

The dentino-enamel junction is not an simple inert interface between two mineralized structures. A less simplistic view suggests that the dentino-enamel junctional complex should also include the inner aprismatic enamel and the mantle dentin. At early stages of enamel formation, fibroblast growth factor (FGF)-2 is stored in and released from the inner aprismatic enamel, possibly under the control of matrix metalloproteinase (MMP)-3. The concentration peak for MMP-2 and -9 observed in the mantle dentin coincided with a very low labeling for TIMP-1 and -2, favoring the cross-talk between mineralizing epithelial and connective structures, and as a consequence the translocation of enamel proteins toward odontoblasts and pulp cells, and vice versa, the translocation of dentin proteins toward secretory ameloblasts and cells of the enamel organ. Finally, in X-linked hypophosphatemic rickets, large interglobular spaces in the circumpulpal dentin were the major defect induced by the gene alteration, whereas the mantle dentin was constantly unaffected. Altogether, these data plead for the recognition of the dentino-enamel junctional complex as a specific entity bearing its own biological characteristics.     

Correlation of the arrangement pattern of enamel rods and secretory ameloblasts in pig and monkey teeth: A possible role of the terminal webs in ameloblast movement during secretion

Enamel rod architecture and ameloblast arrangement were examined in pig and monkey teeth using light microscopy and scanning and transmission electron microscopy. Enamel rods in the pig teeth were arranged in longitudinal straight rows in the initial enamel layer, in longitudinal wavy rows in the inner enamel layer, and in a staggered pattern in the outer enamel layer. Rod decussation was seen only in the inner layer. Cross-sectined enamel rods in the pig were arcade-shaped in the initial and inner layers, and mostly round in shape with circular boundaries in the outer layer. Arrangement of secretory ameloblasts at the level of the distal terminal web and Tomes' processes, and shape of Tomes' processes, corresponded to those of the enamel rod in the enamel layers. Distal terminal webs were well developed between straight rows of the ameloblasts forming the initial layer and between wavy rows of the ameloblasts forming the inner layer, and less developed within a row. The filament bundles in the distal terminal webs were also oriented along the rows. However, in the ameloblasts forming the outer layer, which lost their row pattern, distal terminal web filaments were distributed uniformly at the cell periphery. A similar arrangement of wavy rows of ameloblasts at the level of distal terminal web and Tomes' processes was also seen in monkey teeth.     

Correlation of the arrangement pattern of enamel rods and secretory ameloblasts in pig and monkey teeth: a possible role of the terminal webs in ameloblast movement during secretion.

Enamel rod architecture and ameloblast arrangement were examined in pig and monkey teeth using light microscopy and scanning and transmission electron microscopy. Enamel rods in the pig teeth were arranged in longitudinal straight rows in the initial enamel layer, in longitudinal wavy rows in the inner enamel layer, and in a staggered pattern in the outer enamel layer. Rod decussation was seen only in the inner layer. Cross-sectioned enamel rods in the pig were arcade-shaped in the initial and inner layers, and mostly round in shape with circular boundaries in the outer layer. Arrangement of secretory ameloblasts at the level of the distal terminal web and Tomes' processes, and shape of Tomes' processes, corresponded to those of the enamel rod in the enamel layers. Distal terminal webs were well developed between straight rows of the ameloblasts forming the initial layer and between wavy rows of the ameloblasts forming the inner layer, and less developed within a row. The filament bundles in the distal terminal webs were also oriented along the rows. However, in the ameloblasts forming the outer layer, which lost their row pattern, distal terminal web filaments were distributed uniformly at the cell periphery. A similar arrangement of wavy rows of ameloblasts at the level of distal terminal web and Tomes' processes was also seen in monkey teeth.     

Identification and characterization of human PRICKLE1 and PRICKLE2 genes as well as mouse Prickle1 and Prickle2 genes homologous to Drosophila tissue polarity gene prickle

Drosophila prickle is implicated in tissue polarity or planar polarity. Here, human PRICKLE1 gene corresponding to FLJ31937 cDNA and human PRICKLE2 gene corresponding to DKFZp686D143 cDNA were identified to be homologous to Drosophila prickle gene by using bioinformatics. PRICKLE1 gene was mapped to human chromosome 12p11-q12, and PRICKLE2 gene was mapped to human chromosome 3p14. Mouse Prickle1 and Prickle2 genes were next identified in mouse genome draft sequences NW_000106.1 and NW_000262.1, respectively. Human PRICKLE1, PRICKLE2, Xenopus Prickle, and Drosophila prickle were homologous in the PET domain, three LIM domains, and the C-terminal Prickle homologous (PKH) domain. LMO6 and TESTIN, containing the PET domain and three LIM domains, were found to lack the PKH domain. Therefore, PRICKLE1 and PRICKLE2 rather than LMO6 and TESTIN were found to be human homologs of Drosophila prickle. PRICKLE1 and PRICKLE2 mRNAs were expressed together in brain, eye and testis. PRICKLE1 mRNA was expressed in fetal heart and hematological malignancies, while PRICKLE2 mRNA in fetal brain, adult cartilage, pancreatic islet, gastric cancer with signet-ring cell features, and uterus tumors. Because tissue polarity genes frizzled, dishevelled, flamingo, and Vang are evolutionary and functionary conserved from Drosophila to human, PRICKLE1 and PRICKLE2 might be implicated in the localization of Frizzled and Dishevelled proteins, just like Drosophila prickle. This is the first report on identification and characterization of human PRICKLE1, PRICKLE2, mouse Prickle1, and Prickle2 genes.     

Tight junctions in differentiating ameloblasts and odontoblasts differentially express ZO-1, occludin, and claudin-1 in early odontogenesis of rat molars

Little is known about the expression of associated proteins during the assembly of tight junctions (TJs). We studied the distribution of ZO-1, occludin, and claudin-1 between differentiating ameloblasts and odontoblasts in molar tooth germs from 1- to 3-day-old rats by confocal laser scanning microscopy. Immunoreactivity for ZO-1 was strong at proximal and distal junctional complexes of differentiating ameloblasts, while it was weak and punctuate at the distal region of differentiating odontoblasts. Occludin was immunoreactive at distal and proximal complexes of early differentiating ameloblasts and at distal regions of differentiating odontoblasts. However, in more advanced stages, occludin was only evident at the proximal complex of ameloblasts. Claudin-1 was strongly detected at the proximal complex but it was weak at distal complex of late differentiating ameloblasts. Thus, our results showed that ZO-1, occludin, and claudin-1 are differentially expressed as TJs assemble for regulating polarity and/or paracellular permeability in differentiating ameloblasts and odontoblasts.     

Fine structure of the secretory and nonsecretory ameloblasts in the frog. I. Fine structure of the secretory ameloblasts

Amelogenesis in the tooth germs of the frog Rana pipiens was examined by electron microscopy at different stages of tooth development. Cellular changes in secretory ameloblasts during this process showed many basic similarities to those in mammalian amelogenesis. Amelogenesis can be divided into three stages based on histological criteria such as thickness of enamel and the relative position of the tooth germ within the continuous succession of teeth. These stages are early, transitional and late. The fine structure of the enamel-secreting cells reflects the functional role of these ameloblasts as primarily secretory in the early stage, possibly transporting in the late stage and reorganizing between the two functions in the transitional stage. In early amelogenesis the cell exhibits well-developed granular endoplasmic reticulum, Golgi complex, microtubules, dense granules, smooth and coated vesicles, lysosome-like bodies in supranuclear and distal portions of the cell and mitochondria initially concentrated in the basal part of the cell. Numerous autophagic vacuoles are observed concomitant with the loss of some cell organelles at the transitional stage. During late amelogenesis the ameloblasts exhibit numerous vesicles, granules, convoluted cell membranes, junctional complexes and widely distributed mitochondria. Toward the end of amelogenesis, cells become oriented parallel to the enamel surface and the number of organelles is reduced. Amelogenesis in the frog is an extracellular process and mineralization seems to occur simultaneously with matrix formation.     

Immunohistochemical demonstration of an enamel sheath protein, sheathlin, in odontogenic tumors

Enamel proteins can be useful markers for assessment of the functional differentiation of neoplastic epithelium and the nature of extracellular matrices in odontogenic tumors. In the present study, we examined immunohistochemical localization of sheathlin, a recently cloned enamel sheath protein, in various odontogenic tumors to evaluate functional differentiation of tumor cells and the nature of hyalinous or calcified matrices in odontogenic neoplasms. Distinct immunolocalization of sheathlin was observed in the immature enamel of the tooth germ at the late bell stage. Secretory ameloblasts facing the enamel matrix also showed positive staining in their cytoplasm. Definite localization of sheathlin was demonstrated in the enamel matrix in odontogenic tumors with inductive dental hard tissue formation such as ameloblastic fibroodontomas and odontomas. Immunoexpression of sheathlin was, furthermore, demonstrated in eosinophilic droplets in solid nests of adenomatoid odontogenic tumor (AOT) and ghost cells in the epithelial lining of calcifying odontogenic cyst (COC). In AOT, cells facing the eosinophilic droplets also expressed the protein in their cytoplasm. There was neither intracellular staining for sheathlin in the tumor cells nor extracellular staining in the matrix of ameloblastomas and calcifying epithelial odontogenic tumors. Dentin, dysplastic dentin-like hyaline material and cementum in the tumors examined were negative for sheathlin. These results show that immunodetection of sheathlin is a useful marker for functional differentiation of secretory ameloblasts and enamel matrix, which is often hard to differentiate from other hard tissues in odontogenic tumors. Our findings from the view point of sheathlin expression support that the tumor cells of ameloblastomas do not attain full differentiation into functional ameloblasts. It is very interesting that epithelial cells in odontogenic tumors can differentiate into functional ameloblasts without induction by odontogenic mesenchyme, as shown by immunoexpression of sheathlin in eosinophilic droplets within solid epithelial sheets in AOT and ghost cells in the epithelial lining of COC where inductive participation of mesenchymal cells was most unlikely. Received: 19 May 1999 / Accepted: 27 September 1999     

Immunolocalization of enamel proteins during amelogenesis in the cat

Amelogenesis in the cat has been suggested to closely resemble enamel formation in human teeth. In order to further characterize the sequence of events leading to enamel formation in the cat, the expression and distribution of enamel proteins throughout amelogenisis were examined by postembedding immunocytochemistry using an antibody to mouse amelogenins and the high resolution protein A-gold technique. Enamel proteins were first immunodetected in ameloblasts and in the extracellular matrix during the presecretory stage. Secretory stage ameloblasts showed the most intense cellular reactivity. In these cells, protein synthetic organelles, secretory granules, and large lysosome-like structures were all intensely labeled. Extracellulary, numerous gold particles were observed over enamel and over patches of material found at the baso-lateral surfaces of these ameloblasts. During the early maturation stage, the protein synthetic organelles and secretory granules of ameloblasts still showed some immunoreactivity, although the most conspicuous labeling at this later stage was found over enamel and over material present amoung the extensive apical membrane infoldings of ruffle-ended ameloblasts. Qualitative analysis of lysosome-like elements in ameloblasts suggested that their frequency and immunoreactivity in the maturation stage were relatively lower than in the secretory stage, where some groups of cells often showed numerous large labeled structures. The enamel matrix was intensely labeled at all stages; however, cervical-occlusal and surface-depth gradients were readily apparent by conventional staining and by quantitative analysis of immunolabeling in the late secretory and early maturation stages. These data suggest that the cellular and extracellular distribution of enamel proteins in the cat is generally similar to that reported in other species, although some particularities were observed, perhaps reflecting variation in the timing of developmental parameters. © 1992 Wiley-Liss, Inc.     

Immunolocalization of enamel proteins during amelogenesis in the cat.

Amelogenesis in the cat has been suggested to closely resemble enamel formation in human teeth. In order to further characterize the sequence of events leading to enamel formation in the cat, the expression and distribution of enamel proteins throughout amelogenesis were examined by postembedding immunocytochemistry using an antibody to mouse amelogenins and the high resolution protein A-gold technique. Enamel proteins were first immunodetected in ameloblasts and in the extracellular matrix during the presecretory stage. Secretory stage ameloblasts showed the most intense cellular reactivity. In these cells, protein synthetic organelles, secretory granules, and large lysosome-like structures were all intensely labeled. Extracellularly, numerous gold particles were observed over enamel and over patches of material found at the baso-lateral surfaces of these ameloblasts. During the early maturation stage, the protein synthetic organelles and secretory granules of ameloblasts still showed some immunoreactivity, although the most conspicuous labeling at this later stage was found over enamel and over material present among the extensive apical membrane infoldings of ruffle-ended ameloblasts. Qualitative analysis of lysosome-like elements in ameloblasts suggested that their frequency and immunoreactivity in the maturation stage were relatively lower than in the secretory stage, where some groups of cells often showed numerous large labeled structures. The enamel matrix was intensely labeled at all stages; however, cervical-occlusal and surface-depth gradients were readily apparent by conventional staining and by quantitative analysis of immunolabeling in the late secretory and early maturation stages. These data suggest that the cellular and extracellular distribution of enamel proteins in the cat is generally similar to that reported in other species, although some particularities were observed, perhaps reflecting variation in the timing of developmental parameters.     

The ameloblast movement in rat incisor. L.M., S.E.M. and C.L.S.M. study

The internal epithelium of enamel organ and the below enamel surface during growth of the lower incisor, were examinated in ten Wistar rat 12-27 weeks old and weighing between 150/200 gr, by means of immuno histochemical, light and scanning electron microscopy techniques. Our specimens indicate that during the outer enamel secretion the anti-actin positivity goes from distal terminal web to infra nuclear region of cell body. The results of the present study do not support the active movement hypothesis, conversely they support the Warshawsky (1992) hypothesis, i.e. the distal terminal web permits the maintenance and the assembling of ameloblasts during enamel growth. Hence we do agree with Osborn (1970) who reported that, during secretion, ameloblasts move passively in response to secretory forces.     

Histochemical, ultrastructural, and electron microprobe analytical studies on the localization of calcium in rat incisor ameloblasts at early stage amelogenesis

The enamel organs of rat incisors were separated from the enamel surface and processed for rapid freezing and freeze-substitution. A histochemical stain for calcium (GBHA) of thick Epon sections revealed intense calcium reactions in the secretory ameloblasts, exclusively in the tubulovesicular structures extending throughout their distal cytoplasm. Electron microscopy revealed a thin layer of amorphous material with clusters of electron-dense granules along the distal surface of secretory ameloblasts. In young secretory ameloblasts without typical Tomes' processes, a considerable number of mitochondria were located in the distal cytoplasm and contained numerous electron-dense granules. Similar dense granules as well as fine ribbon-like electron-dense figures, all containing significant amounts of calcium, were observed in some of the tubulovesicular structures at the distal end of these cells. A putative exocytotic figure of such dense granules was also observed. The electron-dense granules were rare in more differentiated ameloblasts with elongated Tomes' processes, which occasionally displayed ribbon-like figures in some of the tubulovesicular structures in the process region. No significant calcium peak was detected in the extracellular amorphous material, secretory granules, or along the lateral plasma membranes. These observations may imply high calcium concentrations in mitochondria and tubulovesicular structures in the distal cytoplasm of secretory ameloblasts relative to that of the cytosol and support the possible contribution of these organelles in secretory ameloblasts to cellular calcium regulation at least in the early stage of amelogenesis.     

Immunohistochemical localization of MMP-2, MMP-9, TIMP-1, and TIMP-2 in the forming rat incisor

Western blots analyses and gelatin zymography established the presence of matrix metalloproteinase (MMP)-2 and -9 in the forming zone of rat incisor. Light microscope immunohistochemistry carried out on undemineralized material provided evidence for strong MMP-2 staining in the secretory ameloblasts, odontoblasts, in the enamel organ, and in the pulp. A weaker staining was observed in predentin and in the outer part of the forming enamel. Using MMP-9 antibodies, the staining was generally weak, except for the secretory ameloblasts that were positively stained. Electron microscopic immunohistochemistry of undemineralized sections revealed a close association between gold-antibodies complexes and cytoskeletal microfilaments in the cytosol of secretory ameloblasts and odontoblasts, within the rough endoplasmic reticulum and along the plasma membrane. The striking feature of MMP-2 and -9 electron immunostaining was the particularly high labeling in the mantle dentin. By contrast, staining of tissue inhibitors of metalloproteinases (TIMP-1 and -2) was lowest in this region. We suggest that this uneven distribution may have some functional implications.     

H+-K+-ATPase activity in the rat incisor enamel organ during enamel formation

The enamel organ of growing rat incisors was perfusion-fixed with a mixture of formaldehyde and glutaraldehyde and processed for ultracytochemical demonstration of ouabain-resistant, K+-stimulated p-nitrophenylphosphatase representing the second dephosphorylative step of H-K-ATPase by use of the one-step lead method. Throughout the stages of amelogenesis, the enzymatic activity was found in the plasma membranes, mitochondrial membranes, and lysosomal structures of the cells of stratum intermedium, papillary layer, and ameloblast layer. Gap junctions and desmosomes between these cells were, however, free of reaction product or showed slight precipitates of reaction. The stellate reticulum and the outer enamel epithelium at the stage of enamel secretion were usually negative for reaction. Although secretory, transition, and ruffle-ended maturation ameloblasts showed enzymatic activity at their basolateral cell surfaces, their distal cell surfaces facing the enamel were always free of reaction product. On the other hand, the smooth-ended maturation ameloblasts seldom showed a positive reaction, except in lysosomes and along their basal cell surfaces. An energy-dispersive X-ray microanalysis of reaction products of H-K-ATPase in unosmicated tissue sections demonstrated that they were composed of lead and phosphorus, which had been released during the dephosphorylation of substrate. In cytochemical controls, the enzymatic activity was completely dependent on substrate and potassium ion, resistant to ouabain and levamisole, and inhibited by nolinium bromide, a specific inhibitor of H-K-ATPase. In addition, inorganic trimetaphosphatase as enzymatic marker of lysosome was localized in dark and pale lysosomes, phagosomes, multivesicular bodies, and ferritin-containing vesicles of the ameloblasts and the cells of stratum intermedium and papillary layer. These membrane-bound structures were also positive for H-K-ATPase reaction. These results suggest that: 1) H-K-ATPase functions to maintain an acidic internal pH of lysosomes in the enamel organ cells; and 2) H-K-ATPase localization in the plasma membranes of enamel organ cells is concerned with efflux of protons derived from cytoplasmic water.     

Immunohistochemical Localization of MMP-2, MMP-9, TIMP-1, and TIMP-2 in the Forming Rat Incisor

Western blots analyses and gelatin zymography established the presence of matrix metalloproteinase (MMP)-2 and -9 in the forming zone of rat incisor. Light microscope immunohistochemistry carried out on undemineralized material provided evidence for strong MMP-2 staining in the secretory ameloblasts, odontoblasts, in the enamel organ, and in the pulp. A weaker staining was observed in predentin and in the outer part of the forming enamel. Using MMP-9 antibodies, the staining was generally weak, except for the secretory ameloblasts that were positively stained. Electron microscopic immunohistochemistry of undemineralized sections revealed a close association between gold-antibodies complexes and cytoskeletal microfilaments in the cytosol of secretory ameloblasts and odontoblasts, within the rough endoplasmic reticulum and along the plasma membrane. The striking feature of MMP-2 and -9 electron immunostaining was the particularly high labeling in the mantle dentin. By contrast, staining of tissue inhibitors of metalloproteinases (TIMP-1 and -2) was lowest in this region. We suggest that this uneven distribution may have some functional implications.