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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.

SUBMITTER: Bori E

PROVIDER: S-EPMC4841280 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:Previous studies have shown that sodium bicarbonate ingestion may enhance intense exercise performance, but may also cause severe gastrointestinal distress. The purpose of this study was to determine whether a modified sodium bicarbonate (SB) ingestion protocol would elevate serum bicarbonate concentration more than previous methods without causing gastrointestinal distress. METHODS:In randomized order, seven (5 men, 2 women) elite middle-distance runners ingested either placebo, Modified SB (600?mg·kg-?1 over 19.5?h), or Acute SB (300?mg·kg-?1) in opaque gelatin capsules. Baseline and post-ingestion blood samples were analyzed for bicarbonate, pH, sodium, hematocrit, and lactate. Repeated measures ANOVA (2 time points ×?3 conditions) were analyzed to determine differences in serum bicarbonate, lactate, sodium, blood pH, and hematocrit. Gastrointestinal distress was assessed via self-report on a Likert scale of 1-10. Simple (condition) and repeated (time) within-participant contrasts were used to determine the location of any statistically significant main and interaction effects (p???0.05). RESULTS:Both Modified SB (7.6?mmol·L-?1, p?<?0.01) and Acute SB (5.8?mmol·L-?1, p?<?0.01) increased serum bicarbonate concentration compared to the placebo (p???0.05). Post-ingestion serum bicarbonate concentration was significantly higher for the Modified SB (34.7?±?2.2?mmol·L-?1, 28.0% increase) trials than the Acute SB (33.5?±?2.0?mmol·L-?1, 20.9% increase) trials (p?=?0.05). There was no reported severe GI distress in the Modified SB trials, but two cases in the Acute SB trials. CONCLUSIONS:Modified SB elevated serum bicarbonate concentration more than Acute SB, without any severe gastrointestinal side effects. Consequently, it is recommended that future experimentation involving SB by researchers and athletes use the novel ingestion protocol described in this study due to its potential for improved effectiveness and reduced gastrointestinal impact. TRIAL REGISTRATION:ClinicalTrials.gov , NCT03813329 . Registered 23 January 2019 - Retrospectively registered.

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Evidence for Bicarbonate Secretion by Ameloblasts in a Novel Cellular Model.

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