Fluctuations in surface pH of maturing rat incisor enamel are a result of cycles of H(+)-secretion by ameloblasts and variations in enamel buffer characteristics.

It is disputed if ameloblasts in the maturation zone of the enamel organ mainly buffer protons released by hydroxyapatite (HA) crystal growth or if they periodically secrete protons to create alternating acidic and alkaline conditions. The latter hypothesis predicts alternating pH regimes in maturing enamel, which would be affected by pharmacological interference with ameloblast H(+)-secretion. This study tests these predictions. Colorimetric pH-indicators and ratiometric fluorometry were used to measure surface pH in maturation zone enamel of rat incisors. Alternating acidic (down to pH6.24±0.06) and alkaline zones (up to pH7.34±0.08) were found along the tooth coinciding with ameloblast morphological cycles. Underlying the cyclic pattern, a gradual decrease in pH towards the incisal edge was seen. Vinblastine or FR167356 (H(+)-ATPase-inhibitor) disturbed ameloblast acid-secretion, especially in the early parts of acidic zones. Enamel surface pH reflects the titration state of surface PO4(3-)-ions. At the pH-values observed, PO4(3-) would be protonated (pKa>12) and HA dissolved. However, by molecular dynamics simulations we estimate the pKa of HPO4(2-) at an ideal HA surface to be 4.3. The acidic pH measured at the enamel surface may thus only dissolve non-perfect domains of HA crystals in which PO4(3-) is less electrostatically shielded. During repeated alkaline/acidic cycles, near-perfect HA-domains may therefore gradually replace less perfect HA-domains resulting in near-perfect HA-crystals. In conclusion, cyclic changes in ameloblast H(+)-secretion and the degree of enamel maturation determine enamel surface pH. This is in accordance with a hypothesis implicating H(+)-ATPase mediated acid-secretion by ameloblasts.

Ion transporters in secretory and cyclically modulating ameloblasts: a new hypothesis for cellular control of preeruptive enamel maturation.

Mature enamel consists of densely packed and highly organized large hydroxyapatite crystals. The molecular machinery responsible for the formation of fully matured enamel is poorly described but appears to involve oscillative pH changes at the enamel surface. We conducted an immunohistochemical investigation of selected transporters and related proteins in the multilayered rat incisor enamel organ. Connexin 43 (Cx-43) is found in papillary cells and ameloblasts, whereas Na(+)-K(+)-ATPase is heavily expressed during maturation in the papillary cell layer only. Given the distribution of Cx-43 channels and Na(+)-K(+)-ATPase, we suggest that ameloblasts and the papillary cell layer act as a functional syncytium. During enamel maturation ameloblasts undergo repetitive cycles of modulation between ruffle-ended (RA) and smooth-ended (SA) ameloblast morphologies. Carbonic anhydrase II and vacuolar H(+)-ATPase are expressed simultaneously at the beginning of the maturation stage in RA cells. The proton pumps are present in the ruffled border of RA and appear to be internalized during the SA stage. Both papillary cells and ameloblasts express plasma membrane acid/base transporters (AE2, NBC, and NHE1). AE2 and NHE1 change position relative to the enamel surface as localization of the tight junctions changes during ameloblast modulation cycles. We suggest that the concerted action of the papillary cell layer and the modulating ameloblasts regulates the enamel microenvironment, resulting in oscillating pH fluctuations. The pH fluctuations at the enamel surface may be required to keep intercrystalline spaces open in the surface layers of the enamel, enabling degraded enamel matrix proteins to be removed while hydroxyapatite crystals grow as a result of influx of calcium and phosphate ions.

Targeted disruption of the Cl-/HCO3- exchanger Ae2 results in osteopetrosis in mice.

Osteoclasts are multinucleated bone-resorbing cells responsible for constant remodeling of bone tissue and for maintaining calcium homeostasis. The osteoclast creates an enclosed space, a lacuna, between their ruffled border membrane and the mineralized bone. They extrude H(+) and Cl(-) into these lacunae by the combined action of vesicular H(+)-ATPases and ClC-7 exchangers to dissolve the hydroxyapatite of bone matrix. Along with intracellular production of H(+) and HCO(3)(-) by carbonic anhydrase II, the H(+)-ATPases and ClC-7 exchangers seems prerequisite for bone resorption, because genetic disruption of either of these proteins leads to osteopetrosis. We aimed to complete the molecular model for lacunar acidification, hypothesizing that a HCO(3)(-) extruding and Cl(-) loading anion exchange protein (Ae) would be necessary to sustain bone resorption. The Ae proteins can provide both intracellular pH neutrality and serve as cellular entry mechanism for Cl(-) during bone resorption. Immunohistochemistry revealed that Ae2 is exclusively expressed at the contra-lacunar plasma membrane domain of mouse osteoclast. Severe osteopetrosis was encountered in Ae2 knockout (Ae2-/-) mice where the skeletal development was impaired with a higher diffuse radio-density on x-ray examination and the bone marrow cavity was occupied by irregular bone speculae. Furthermore, osteoclasts in Ae2-/- mice were dramatically enlarged and fail to form the normal ruffled border facing the lacunae. Thus, Ae2 is likely to be an essential component of the bone resorption mechanism in osteoclasts.