Functional and Molecular Characterization of Transmembrane Intracellular pH Regulators in Human Dental Pulp Stem Cells

Publication date: Available online 6 March 2018
Source:Archives of Oral Biology
Author(s): Gunng-Shinng Chen, Shiao-Pieng Lee, Shu-Fu Huang, Shih-Chi Chao, Chung-Yi Chang, Gwo-Jang Wu, Chung-Hsing Li, Shih-Hurng Loh
ObjectiveHomeostasis of intracellular pH (pHi) plays vital roles in many cell functions, such as proliferation, apoptosis, differentiation and metastasis. Thus far, Na⁺-H⁺ exchanger (NHE), Na⁺-HCO3⁻ co-transporter (NBC), Cl⁻/HCO3⁻ exchanger (AE) and Cl⁻/OH⁻ exchanger (CHE) have been identified to co-regulate pHi homeostasis. However, functional and biological pHi-regulators in human dental pulp stem cells (hDPSCs) have yet to be identified.DesignMicrospectrofluorimetry technique with pH-sensitive fluorescent dye, BCECF, was used to detect pHi changes. NH4Cl and Na⁺-acetate pre-pulse were used to induce intracellular acidosis and alkalosis, respectively. Isoforms of pHi-regulators were detected by Western blot technique.ResultsThe resting pHi was no significant difference between that in HEPES-buffered (nominal HCO3⁻-free) solution or CO2/HCO3-buffered system (7.42 and 7.46, respectively). The pHi recovery following the induced-intracellular acidosis was blocked completely by removing [Na⁺]o, while only slowed (-63%) by adding HOE694 (a NHE1 specific inhibitor) in HEPES-buffered solution. The pHi recovery was inhibited entirely by removing [Na⁺]o, while adding HOE 694 pulse DIDS (an anion-transporter inhibitor) only slowed (-55%) the acid extrusion. Both in HEPES-buffered and CO2/HCO3-buffered system solution, the pHi recovery after induced-intracellular alkalosis was entirely blocked by removing [Cl⁻]o. Western blot analysis showed the isoforms of pHi regulators, including NHE1/2, NBCe1/n1, AE1/2/3/4 and CHE in the hDPSCs.ConclusionsWe demonstrate for the first time that resting pHi is significantly higher than 7.2 and meditates functionally by two Na⁺-dependent acid extruders (NHE and NBC), two Cl⁻-dependent acid loaders (CHE and AE) and one Na⁺-independent acid extruder(s) in hDPSCs. These findings provide novel insight for basic and clinical treatment of dentistry.



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