title: 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 Ca(2+)- 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.
authors.affiliation: Department of Oral Biology, Semmelweis University, Budapest, Hungary.
authors.forename: E
authors.initials: E
authors.lastname: Bori
authors.affiliation: Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam, University of Amsterdam and VU University Amsterdam, MOVE Research Institute, Amsterdam, Netherlands.
authors.forename: J
authors.initials: J
authors.lastname: Guo
authors.affiliation: Department of Oral Biology, Semmelweis University, Budapest, Hungary.
authors.forename: R
authors.initials: R
authors.lastname: Rácz
authors.affiliation: Department of Oral Biology, Semmelweis University, Budapest, Hungary.
authors.forename: B
authors.initials: B
authors.lastname: Burghardt
authors.affiliation: Department of Oral Biology, Semmelweis University, Budapest, Hungary.
authors.forename: A
authors.initials: A
authors.lastname: Földes
authors.affiliation: Department of Oral Biology, Semmelweis University, Budapest, Hungary.
authors.forename: B
authors.initials: B
authors.lastname: Kerémi
authors.affiliation: Department of Anatomy, Division of Developmental Biology and Regenerative Medicine, Iwate Medical University, Iwate, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Harada
authors.affiliation: Faculty of Life Sciences, The University of Manchester, Manchester, UK.
authors.forename: M C
authors.initials: MC
authors.lastname: Steward
authors.affiliation: Department of Orofacial Sciences, University of California, San Francisco, CA, USA.
authors.forename: P
authors.initials: P
authors.lastname: Den Besten
authors.affiliation: Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam, University of Amsterdam and VU University Amsterdam, MOVE Research Institute, Amsterdam, Netherlands.
authors.forename: A L J J
authors.initials: AL
authors.lastname: Bronckers
authors.affiliation: Department of Oral Biology, Semmelweis University, Budapest, Hungary varga.gabor@dent.semmelweis-univ.hu.
authors.forename: G
authors.initials: G
authors.lastname: Varga
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors; D000086:Acetazolamide / Q000494:pharmacology; D000565:Ameloblasts / Q000378:metabolism*; D000818:Animals; D001639:Bicarbonates / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D002245:Carbon Dioxide / Q000378:metabolism; D002257:Carbonic Anhydrase Inhibitors / Q000494:pharmacology; D002352:Carrier Proteins / Q000032:analysis; D018929:Cell Culture Techniques; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000502:physiology; D016764:Cell Polarity / Q000502:physiology; D062445:Claudin-1 / Q000032:analysis; D062506:Claudin-4 / Q000032:analysis; D057167:Claudins / Q000032:analysis; D000242:Cyclic AMP / Q000494:pharmacology; D003746:Dental Enamel Proteins / Q000032:analysis; D017097:Electric Impedance; D005470:Fluorometry / Q000379:methods; D006863:Hydrogen-Ion Concentration; D007610:Kallikreins / Q000032:analysis; D051381:Rats; D012964:Sodium / Q000494:pharmacology; D019108:Tight Junctions / Q000187:drug effects / Q000502:physiology
chemical_list: C514056:Amtn protein, rat; D001639:Bicarbonates; D002257:Carbonic Anhydrase Inhibitors; D002352:Carrier Proteins; D062445:Claudin-1; D062506:Claudin-4; D057167:Claudins; D003746:Dental Enamel Proteins; D002245:Carbon Dioxide; C018564:dihydro-DIDS; D012964:Sodium; D000242:Cyclic AMP; D007610:Kallikreins; C120801:kallikrein 4; C502011:claudin 8; D000086:Acetazolamide; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D002118:Calcium
keywords: cytophotometry; dental enamel; fluorescent dyes; in vitro techniques; ion transport; tissue engineering