CELLULAR CONTROLS OF ENAMEL FLUID COMPOSITION AND PH

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The long-term objective of this research is to define the driving force for mineralization in developing enamel and to understand how it is maintained by cells of the enamel organ, especially the ameloblasts. Of particular interest is the question of how enamel organ cells neutralize excessive hydrogen ions released as new unit cells of hydroxyapatite grow in cyclic bursts at the surfaces of maturing crystals. Our working hypothesis is that bicarbonate ions, generated by carbonic anhydrase, are secreted from the apical surfaces of ameloblasts when they are ruffle-ended perhaps by a mechanism involving a sodium-independent chloride-bicarbonate anti-porter similar to the band 3 protein in erythrocytes. It is further hypothesized that ruffle-ended ameloblasts may also use a sodium-dependent chloride-bicarbonate symporter to exchange intracellular hydrochloric acid for extracellular sodium bicarbonate at their basolateral surfaces. Periodic inability to maintain pH balance and/or ionic composition of enamel fluid could account for some of the developmental abnormalities seen in enamel. There are 4 specific aims to this project. Aim 1 is to determine the extent to which isolated pieces of free-dried enamel will mature when exposed to solutions of defined pH and ionic composition and, as required, supplemented with recombinant/native enamel proteins and/or proteinases or natural inhibitors. Aim 2 is to estimate the size (volume), pH and exact ionic composition of the fluid bathing maturing enamel in vivo. Aim 3 is to characterize the main membrane ion transport systems that enamel organ cells use to maintain ionic composition and pH of the extracellular enamel fluid as per Aim 2. Lastly, Aim 4 is to characterize the various isoforms of carbonic anhydrase that are present in enamel organ cells, especially ameloblasts, and determine by inhibitor studies if this enzyme is crucial to the mineralization process. The well-characterized rodent incisor tooth will be used as a model system for these studies that will involve direct 3d-LM imaging, TEM and SEM imaging, as well confocal and direct 3D fluorescence imaging.