SUMMARYMineralized tissues are unique in using proteins to attract and organize calcium and phosphate ions into a structured mineral phase. A precise knowledge of the expression and extracellular distribution of matrix proteins is therefore very important in understanding their function. The purpose of this investigation was to obtain comparative information on the expression, intracellular and extracellular distribution, and dynamics of proteins representative of the two main classes of enamel matrix proteins. Amelogenins were visualized using an antibody and an mRNA probe prepared against the major alternatively spliced isoform in rodents, and nonamelogenins by antibodies and mRNA probes specific to one enamel protein referred to by three names: ameloblastin, amelin, and sheathlin. Qualitative and quantitative immunocytochemistry, in combination with immunoblotting and in situ hybridization, indicated a correlation between mRNA signal and sites of protein secretion for amelogenin, but not for ameloblastin, during the early presecretory and midto late maturation stages, during which mRNA signals were detected but no proteins appeared to be secreted. Extracellular amelogenin immunoreactivity was generally weak near secretory surfaces, increasing over a distance of about 1.25 m to reach a level slightly above an amount expected if the protein were being deposited evenly across the enamel layer. Immunolabeling for ameloblastin showed an inverse pattern, with relatively more gold particles near secretory surfaces and much fewer deeper into the enamel layer. Administration of brefeldin A and cycloheximide to stop protein secretion revealed that the immunoblotting pattern of amelogenin was relatively stable, whereas ameloblastin broke down rapidly into lower molecular weight fragments. The distance from the cell surface at which immunolabeling for amelogenin stabilized generally corresponded to the point at which that for ameloblastin started to show a net reduction. These data suggest a correlation between the distribution of amelogenin and ameloblastin and that intact ameloblastin has a transient role in promoting/stabilizing crystal elongation. A meloblasts , like all hard tissue-forming cells, release an intricate set of extracellular matrix proteins optimized for promoting the development of a closely associated mineral phase (reviewed in Deutsch et al.

The granular convoluted tubule segment (GCT) of the rodent submandibular gland is a unique structure and consists of granular cell, dark granular cell, pillar cell, and transitional cell. The granular cells contain secretory granules and synthesize biologically active peptides and related processing enzymes; EGF, NGF, kallikrein and renin, erythropoietin, atrial natriuretic peptides, large mobile protein as cystain and serin proteinase inhibitors, S-100 protein, glucagon and somatostatin.Granular cells may have a multiproduction system for growth factors or biologically active peptides for related processing proteinases as well as proteinase inhibitors which are secreted by a-adrenergic stimulants. Pillar cells contain S-100 protein, neuron specific enolase and other nerve related materials, and involve regulation of the nerverelated system as neurotransmitter-like or paraneuron-like properties. Transitional cells show combined properties of granular convoluted tubule and striated duct, and may participate in secretion and reabsoorption. Granular intercalated duct cells show similar category of granular cells in GCT segment.

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S U M M A R YDuring the maturation stage of amelogenesis, the loss of matrix proteins combined with an accentuated but regulated influx of calcium and phosphate ions into the enamel layer results in the "hardest" tissue of the body. The aim of the present investigation was to examine the effects of chronic hypocalcemia on the maturation of enamel. Twenty-one-day old male Wistar rats were given a calcium-free diet and deionized water for 28 days, while control animals received a normal chow. The rats were perfused with aldehyde and the mandibular incisors were processed for histochemical and ultrastructural analyses and for postembedding colloidal gold immunolabeling with antibodies to amelogenin, ameloblastin, and albumin. The maturation stage enamel organ in hypocalcemic rats exhibited areas with an apparent increase in cell number and the presence of cyst-like structures. In both cases the cells expressed signals for ameloblastin and amelogenin. The content of the cysts was periodic acid-Schiff-and periodic acid-silver nitrate-methanamine-positive and immunolabeled for amelogenin, ameloblastin, and albumin. Masses of a similar material were also found at the enamel surface in depressions of the ameloblast layer. In addition, there were accumulations of glycoproteinaceous matrix at the interface between ameloblasts and enamel. In decalcified specimens, the superficial portion of the enamel matrix sometimes exhibited the presence of tubular crystal "ghosts." The basal lamina, normally separating ameloblasts and enamel during the maturation stage, was missing in some areas. Enamel crystals extended within membrane invaginations at the apical surface of ameloblasts in these areas. Immunolabeling for amelogenin, ameloblastin, and albumin over enamel was variable and showed a heterogeneous distribution. In contrast, enamel in control rats exhibited a homogeneous labeling for amelogenin, a concentration of ameloblastin at the surface, and weak reactivity for albumin. These results suggest that diet-induced chronic hypocalcemia interferes with both cellular and extracellular events during enamel maturation.

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