Functional and molecular characterization of transmembrane intracellular pH regulators in human dental pulp stem cells

Abstract

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.