CELLULAR CONTROLS OF ENAMEL FLUID COMPOSITION AND PH

这项研究的长期目标是确定釉质发育过程中矿化的驱动力，并了解釉质器官的细胞，特别是成釉细胞是如何维持矿化的。尤其令人感兴趣的问题是，釉质器官细胞如何中和当羟基磷灰石的新单位细胞在成熟晶体表面循环爆发生长时释放出的过量氢离子。我们的工作假设是，碳酸氢酶产生的碳酸氢根离子，当成釉细胞被褶皱结束时，从它们的顶端表面分泌出来，可能是由于一种与红细胞中带3蛋白类似的钠非依赖的氯-碳酸氢盐反转运蛋白的机制。进一步推测，皱褶端成釉细胞也可能利用钠依赖的氯-碳酸氢盐共转运体，在其基底外侧表面用细胞内的盐酸交换细胞外的碳酸氢钠。周期性地不能维持牙釉质液体的pH平衡和/或离子组成可能是牙釉质发育异常的部分原因。这个项目有4个具体目标。目标1是确定游离干燥的釉质在暴露于特定pH和离子组成的溶液中时的成熟程度，并根据需要添加重组/天然釉质蛋白和/或蛋白酶或天然抑制剂。目的2是估计在体液浴成熟牙釉质的大小(体积)、pH和准确的离子组成。目标3是根据目标2描述釉器细胞用来维持细胞外液离子组成和pH的主要膜离子转运系统。最后，目标4是表征釉器细胞，特别是成釉细胞中存在的碳酸酐酶的各种异构体，并通过抑制剂研究该酶是否在矿化过程中起关键作用。具有良好特性的啮齿动物门牙将被用作这些研究的模型系统，包括直接3D-LM成像、透射电子显微镜和扫描电子显微镜成像，以及共焦和直接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.