IADR Abstract Archives
Differential Basolateral and Apical AE and NKCC Activities Demonstrate Polarization of HAT-7 Ameloblasts
Objectives: A coordinated transport of chloride and bicarbonate ions is required for enamel maturation during amelogenesis. We developed a two dimensional cellular model to investigate ion transport mechanisms using the HAT-7 rat ameloblast cell line (Racz et al, Frontiers Physiol., 2018). In the present study we compared the apical and basolateral HCO3-/Cl- anion-exchanger (AE) and sodium-potassium-chloride cotransporter (NKCC) activities in HAT-7 cells.
Methods:
To obtain monolayers, HAT-7 cells were cultured in differentiation medium for 4 days on Transwell membranes. We monitored transepithelial resistance as an indicator of tight junction formation and polarization. We evaluated intracellular pH changes by microfluorometry using BCECF fluorochrome. The activity of anion exchangers was tested by withdrawal of chloride-ions, and using anion exchange inhibitor DIDS. NKCC activity was monitored by the ammonium-pulse technique, after a rapid intracellular alkalization using the specific NKCC inhibitor bumetanide.
Results: In HAT-7 cells grown on Transwell, the basolateral AE activity was high, and could be inhibited by DIDS (0.149±0.017 vs 0.055±0.008 dpH/dt, p<0.05). On the other hand, AE activity was very low at the apical side, and not significantly affected by DIDS (0.020±0.009 vs -0.006±0.006 dpH/dt, p>0.05). Additionally, we found a substantial NKCC activity in HAT-7 cells indicated by the rapid decrease of intracellular pH after an alkaline load (-0.084±0.005 dpH/dt). This activity was substantially inhibited by basolateral bumetanide administration (-0.060±0.004 dpH/dt, p<0.05), but not by apical bumetanide (-0.068±0.004 dpH/dt, p>0.05), indicating the presence of NKCC activity only at the basolateral but not at the apical side of the ameloblast originated cells.
Conclusions: We identified differentially localized activities of AE and NKCC transporters in HAT-7 ameloblast cells, polarized on Transwell membranes. The side-specific activities of these transporters demonstrate that the HAT-7 model is useful to conduct functional studies on ion transport mechanisms of amelogenesis. Supported by EFOP-3.6.2.-16-2017-0006 and NKFIH K-125161.
Methods:
To obtain monolayers, HAT-7 cells were cultured in differentiation medium for 4 days on Transwell membranes. We monitored transepithelial resistance as an indicator of tight junction formation and polarization. We evaluated intracellular pH changes by microfluorometry using BCECF fluorochrome. The activity of anion exchangers was tested by withdrawal of chloride-ions, and using anion exchange inhibitor DIDS. NKCC activity was monitored by the ammonium-pulse technique, after a rapid intracellular alkalization using the specific NKCC inhibitor bumetanide.
Results: In HAT-7 cells grown on Transwell, the basolateral AE activity was high, and could be inhibited by DIDS (0.149±0.017 vs 0.055±0.008 dpH/dt, p<0.05). On the other hand, AE activity was very low at the apical side, and not significantly affected by DIDS (0.020±0.009 vs -0.006±0.006 dpH/dt, p>0.05). Additionally, we found a substantial NKCC activity in HAT-7 cells indicated by the rapid decrease of intracellular pH after an alkaline load (-0.084±0.005 dpH/dt). This activity was substantially inhibited by basolateral bumetanide administration (-0.060±0.004 dpH/dt, p<0.05), but not by apical bumetanide (-0.068±0.004 dpH/dt, p>0.05), indicating the presence of NKCC activity only at the basolateral but not at the apical side of the ameloblast originated cells.
Conclusions: We identified differentially localized activities of AE and NKCC transporters in HAT-7 ameloblast cells, polarized on Transwell membranes. The side-specific activities of these transporters demonstrate that the HAT-7 model is useful to conduct functional studies on ion transport mechanisms of amelogenesis. Supported by EFOP-3.6.2.-16-2017-0006 and NKFIH K-125161.