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

Objective: Homeostasis of intracellular pH (pH;) 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 pH, homeostasis. However, functional and biological pH 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 pH(i) changes. NH4Cl and Na+-acetate pre-pulse were used to induce intracellular acidosis and alkalosis, respectively. Isoforms of pH(i)-regulators were detected by Western blot technique. Results: The resting pH, 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 pH, 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 pH, 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 pH, recovery after induced intracellular alkalosis was entirely blocked by removing [Cl-](o). Western blot analysis showed the isoforms of pH, regulators, including NHE1/2, NBCe1/n1, AE1/2/3/4 and CHE in the hDPSCs. Conclusions: We demonstrate for the first time that resting pH, 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.