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

Homeostasis of intracellular pH (pH_i) 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_i homeostasis. However, functional and biological pH_i-regulators in human dental pulp stem cells (hDPSCs) have yet to be identified.

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.

The resting pH_i 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_i 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_i 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_i recovery after induced-intracellular alkalosis was entirely blocked by removing [Cl^-]_o. Western blot analysis showed the isoforms of pH_i regulators, including NHE1/2, NBCe1/n1, AE1/2/3/4 and CHE in the hDPSCs.

We demonstrate for the first time that resting pH_i 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.