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. NH4 Cl 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.