CELLULAR CONTROLS OF ENAMEL FLUID COMPOSITION AND PH

本研究的长期目标是确定釉质发育中矿化的驱动力，并了解成釉器官细胞，特别是成釉细胞如何维持矿化。特别令人感兴趣的是，釉质器官细胞如何中和在成熟晶体表面的羟基磷灰石新单位细胞周期性爆发生长时释放的过量氢离子的问题。我们的工作假设是，碳酸氢根离子，产生的碳酸酐酶，分泌从成釉细胞的顶端表面时，他们是皱褶结束，也许是由一个机制，涉及钠独立的氯-碳酸氢盐反向转运类似的带3蛋白在红细胞。进一步假设皱褶端成釉细胞也可能使用钠依赖性氯-碳酸氢盐同向转运体在其基底外侧表面将细胞内盐酸交换为细胞外碳酸氢钠。周期性的不能维持pH值平衡和/或离子组成的釉质液可以解释一些发育异常中看到的釉质。该项目有四个具体目标。目的1是确定当暴露于具有确定的pH和离子组成的溶液时，分离的自由干燥的釉质片将成熟的程度，并且根据需要，补充有重组/天然釉质蛋白和/或蛋白酶或天然抑制剂。目的2是估计在体内浸泡成熟釉质的流体的尺寸（体积）、pH和确切的离子组成。目的3是表征成釉器官细胞用于维持细胞外釉质液的离子组成和pH的主要膜离子转运系统，如目的2所述。最后，目的4是表征成釉器官细胞，特别是成釉细胞中存在的碳酸酐酶的各种亚型，并通过抑制剂研究确定这种酶是否对矿化过程至关重要。良好表征的啮齿动物切牙将用作这些研究的模型系统，这些研究将涉及直接3d-LM成像，TEM和SEM成像，以及共聚焦和直接3D荧光成像。

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.