Evidence for Bicarbonate Secretion by Ameloblasts in a Novel Cellular Model

Abstract

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.