Abstract
We have recently developed a novel in vitro model using HAT-7 rat ameloblast cells to functionally study epithelial ion transport during amelogenesis. Our present aims were to identify key transporters of bicarbonate in HAT-7 cells and also to examine the effects of fluoride exposure on vectorial bicarbonate transport, cell viability, and the development of transepithelial resistance. To obtain monolayers, the HAT-7 cells were cultured on Transwell permeable filters. We monitored transepithelial resistance (TER) as an indicator of tight junction formation and polarization. We evaluated intracellular pH changes by microfluorometry using the fluorescent indicator BCECF. Activities of ion transporters were tested by withdrawal of various ions from the bathing medium, by using transporter specific inhibitors, and by activation of transporters with forskolin and ATP. Cell survival was estimated by alamarBlue assay. Changes in gene expression were monitored by qPCR. We identified the activity of several ion transporters, NBCe1, NHE1, NKCC1, and AE2, which are involved in intracellular pH regulation and vectorial bicarbonate and chloride transport. Bicarbonate secretion by HAT-7 cells was not affected by acute fluoride exposure over a wide range of concentrations. However, tight-junction formation was inhibited by 1 mM fluoride, a concentration which did not substantially reduce cell viability, suggesting an effect of fluoride on paracellular permeability and tight-junction formation. Cell viability was only reduced by prolonged exposure to fluoride concentrations greater than 1 mM. In conclusion, cultured HAT-7 cells are functionally polarized and are able to transport bicarbonate ions from the basolateral to the apical fluid spaces. Exposure to 1 mM fluoride has little effect on bicarbonate secretion or cell viability but delays tight-junction formation, suggesting a novel mechanism that may contribute to dental fluorosis.
Highlights
Dental enamel is the hardest material in the human body and its mineral concentration is the highest
In our previous work we showed data suggesting the existence of vectorial, basolateral-to-apical bicarbonate transport in HAT7 ameloblast cells but we did not identify the individual transporters at the basolateral side (Bori et al, 2016)
Na+ restoration on the basolateral side caused a rapid recovery of pHi which was sensitive to 300 μM amiloride (Figures 1A,B) indicating the existence of basolateral Na+-H+ exchanger (NHE) activity, most probably due to NHE1 which is ubiquitously expressed at the basolateral membrane of secretory epithelia
Summary
Dental enamel is the hardest material in the human body and its mineral concentration is the highest. Its major disorders result from either environmental or genetic conditions. In both cases mineral formation can be greatly impaired. Dental caries and erosion are important enamel-loss conditions where reconstruction would be the optimal solution. Ameloblasts secrete enamel in a two-stage process. The remodeling of this matrix results in a high level of mineralization (Robinson, 2014). Ameloblasts have epithelial tight junctions which close the intercellular space allowing the preservation of great concentration gradients between the apical and basal sides of the cells. Calcium and phosphate ions are actively transported into the mineralization space by an only partially understood process
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
