FIG. 2.

Major mechanisms of osteoblast acid transport that support complete conversion of phosphate and calcium to hydroxyapatite. Osteoblasts actively transport phosphate,²³ which is converted from pyrophosphate or nucleotide phosphates by alkaline phosphatase and other phosphatases. Hydroxyapatite is organized, in part, by accessory proteins, but the process of crystallization generates massive amounts of acid. Removal of this acid has the effect of driving precipitation to completion, regardless of extracellular conditions. (A) Key mechanisms driving bone mineral formation to completion include uptake of acid by ClC exchangers at the apical membrane, and acid transcytosis with elimination by NHE exchangers at the basolateral membrane. Modified from,⁵ with permission. (B) Demonstration of massive sodium–hydrogen exchange capacity. Osteoblasts exposed to a large acid load, 30 mM propionic acid, a membrane-permeant organic acid, maintain their internal pH at neutral in the presence of sodium. In the absence of sodium, the cells acidify dramatically. The acid resistance is due to two highly expressed sodium–hydrogen exchangers on the basolateral membranes of osteoblasts. Modified from Liu et al.,²¹ with permission. (C) Human osteoblasts express the chloride-hydrogen exchanger ClC3 at high levels. Fluorescent antibody labeling of ClC3 in bone cortex is shown; the osteoblastic surface is at top and there is very high expression of ClC3 in the apical membranes. The diagram at right shows cells and processes, the labeling of which is demonstrated in the photograph. ClC3 occurs also in the canalicular system. ClC3-knockout controls were unlabeled (not shown). If ClC3 is knocked out, ClC5 expression increases and has the same pattern of expression (not shown). Modified from Larrouture et al.,⁵ with permission. (D) Bone formation by wild-type and ClC3/5-null osteoblasts. Wild-type murine osteoblasts make bone in characteristic nodules with very strong mineral deposition or alkaline phosphatase activity (left panels). Osteoblasts express two ClCs, 3 and 5, on their apical membranes. When ClC3 is knocked out, there is a minor bone phenotype (not shown).⁵ When both ClC3 and 5 are eliminated, there is no mineralization (top right). The lack of ClC expression does not affect alkaline phosphatase expression (bottom). Two centimeter diameter cultures, in six-well plates, of murine osteoblasts at 14 days in the osteoblast medium without cortisol. Modified from Larrouture et al.,⁵ with permission.