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

OBJECTIVE: Homeostasis 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.

DESIGN: Microspectrofluorimetry 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.

RESULTS: The 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.

CONCLUSIONS: We 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.