Evidence for Bicarbonate Secretion by Ameloblasts in a Novel Cellular Model

Formation and growth of hydroxyapatite crystals during amelogenesis generate a large number of protons that must be neutralized,presumably by hco3 - ions transported from ameloblasts into the developing enamel matrix. ameloblasts express a number of transporters and channels known to be involved in hco3 - transport in other epithelia. however,to date,there is no functional evidence for hco3 - transport in these cells. to address questions related to hco3 - export from ameloblasts,we have developed a polarized 2-dimensional culture system for hat-7 cells,a rat cell line of ameloblast origin. hat-7 cells were seeded onto transwell permeable filters. transepithelial resistance was measured as a function of time,and the expression of transporters and tight junction proteins was investigated by conventional and quantitative reverse transcription polymerase chain reaction. intracellular ph regulation and hco3 - transport were assessed by microfluorometry. hat-7 cells formed epithelial layers with measureable transepithelial resistance on transwell permeable supports and expressed claudin-1,claudin-4,and claudin-8 - key proteins for tight junction formation. transport proteins previously described in maturation ameloblasts were also present in hat-7 cells. microfluorometry showed that the hat-7 cells were polarized with a high apical membrane co2 permeability and vigorous basolateral hco3 - uptake,which was sensitive to na+ withdrawal,to the carbonic anhydrase inhibitor acetazolamide and to h2dids inhibition. measurements of transepithelial hco3 - transport showed a marked increase in response to ca2+- and camp-mobilizing stimuli. collectively,2-dimensional hat-7 cell cultures on permeable supports 1) form tight junctions,2) express typical tight junction proteins and electrolyte transporters,3) are functionally polarized,and 4) can accumulate hco3 - ions from the basolateral side and secrete them at the apical membrane. these studies provide evidence for a regulated,vectorial,basolateral-to-apical bicarbonate transport in polarized hat-7 cells. we therefore propose that the hat-7 cell line is a useful functional model for studying electrolyte transport by ameloblasts. © 2016 international & american associations for dental research.