CELLULAR CONTROLS OF ENAMEL FLUID COMPOSITION AND PH

本研究的长期目标是确定牙釉质发育过程中矿化的驱动力，并了解牙釉质器官的细胞，特别是成釉细胞如何维持矿化。特别令人感兴趣的问题是珐琅器官细胞如何中和过量的氢离子，这些氢离子是由成熟晶体表面的羟基磷灰石新单位细胞在循环爆发中生长时释放的。我们的工作假设是，由碳酸酐酶产生的碳酸氢盐离子在成釉细胞卷曲时从其顶端表面分泌，可能是通过一种类似于红细胞中带3蛋白的不依赖钠的氯化物-碳酸氢盐抗转运蛋白的机制。进一步假设，褶边端成釉细胞也可能使用钠依赖的氯化物-碳酸氢钠同质体在其基底外侧表面将细胞内的盐酸交换为细胞外的碳酸氢钠。周期性地不能维持pH值平衡和/或牙釉质液的离子组成可以解释在牙釉质中看到的一些发育异常。这个项目有4个具体目标。目的1是确定分离的游离干牙釉质在暴露于规定pH值和离子组成的溶液中，并根据需要补充重组/天然牙釉质蛋白和/或蛋白酶或天然抑制剂时的成熟程度。目的二是估计体内浸泡成熟牙釉质的液体的大小（体积）、pH值和确切的离子组成。目的3是根据目的2描述牙釉质器官细胞用来维持细胞外牙釉质液的离子组成和pH值的主要膜离子运输系统。最后，目的4是表征存在于牙釉质器官细胞，特别是成釉细胞中的碳酸酐酶的各种异构体，并通过抑制剂研究确定该酶是否对矿化过程至关重要。表征良好的啮齿动物门牙将被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.