Importance of bicarbonate transport in pH control during amelogenesis - need for functional studies

Abstract

Dental enamel, the hardest mammalian tissue, is produced by ameloblasts. Ameloblasts show many similarities to other transporting epithelia although their secretory product, the enamel matrix, is quite different. Ameloblasts direct the formation of hydroxyapatite crystals, which liberate large quantities of protons that then need to be buffered to allow mineralization to proceed. Buffering requires a tight pH regulation and secretion of bicarbonate by ameloblasts. Many investigations have used immunohistochemical and knockout studies to determine the effects of these genes on enamel formation, but up till recently very little functional data were available for mineral ion transport. To address this, we developed a novel 2D in vitro model using HAT-7 ameloblast cells. HAT-7 cells can be polarized and develop functional tight junctions. Furthermore, they are able to accumulate bicarbonate ions from the basolateral to the apical fluid spaces. We propose that in the future, the HAT-7 2D system along with similar cellular models will be useful to functionally model ion transport processes during amelogenesis. Additionally, we also suggest that similar approaches will allow a better understanding of the regulation of the cycling process in maturation-stage ameloblasts, and the pH sensory mechanisms, which are required to develop sound, healthy enamel.

Keywords: HAT-7; ameloblast; amelogenesis; bicarbonate; enamel; functional model; in vitro; ion transport; microfluorometry; pH regulation; transepithelial resistance; transwell.

Figure 1. pH cycle in the luminal surface of ameloblasts during the maturation phase of amelogenesis

Cells cycle between smooth-ended and ruffle-ended stages multiple times. This cycling is accompanied by luminal pH changes between approximately 6.2 and 7.2. The primary driver of acidification is the release of protons liberated during hydroxyl-apatite formation. Acidification is counterbalanced then by the buffering effect of bicarbonate.

Figure 2. Schematic representation of the 2D HAT-7 cellular model, and the method for assessing vectorial, basolateral-to-apical bicarbonate transport in this system

HAT-7 cells, grown on Transwell support, form a tight epithelium, and are polarized to show distinct apical and basolateral surfaces. Vectorial bicarbonate transport is tested in this system by the pH drop method using microfluorometry. As transporters involved at the basolateral side involved in bicarbonate uptake and proton extrusion are blocked by specific inhibitors H₂DIDS and amiloride, respectively, and apical bicarbonate loss is accelerated by secretagogue stimulation, a sudden decrease of intracellular pH can be detected. By appropriate calibrations, the apical base flux, ie bicarbonate extrusion, can be quantitatively estimated.

Figure 3. Schematic drawing of the proposed mechanism of vectorial bicarbonate transport of HAT-7 cells, showing the major ion channels and transporters involved

For most transporters, specific inhibitors or activators were used, as shown on the figure, to test their involvement in intracellular pH control, bicarbonate and Cl- secretion. Note that data about the H⁺-ATPase are shown based on in vivo studies using its specific inhibitor FR167356, but no molecular physiology studies were applied to test its direct involvement in maturation-associated ameloblast cell transport processes.