Abstract
The authors used isoform-specific antibodies against cation (NHE) and anion (AE) exchange isoforms in order to establish their specific expression and localization in dispersed human bone-derived cells. Immunocytochemical preparations of permeabilized osteoblasts probed with polyclonal antibodies were optically analysed by conventional immunofluorescence and con-focal laser scanning microscopy. These techniques demonstrated the abundant presence of epitopes of the cation exchangers NHE1 and NHE3 and the anion exchanger AE2 in these cells. The NHE1 and NHE3 isoform proteins were predominantly located in subplasmalemmal and nucleoplasmic vesicles. The AE2 isoform was densely localized to a subcellular location characteristic of the Golgi complex. The molecular identity of the AE and NHE isoforms was investigated by RT-PCR that confirmed the presence of NHE1 and NHE3 transcripts in addition to NHE4. RT-PCR and diagnostic restriction analysis of amplified AE cDNA established preferential AE2 expression. Since AE2 has been shown to act as a sulfate transporter at low pH, it is possible that it performs this function in the osteoblast Golgi complex where sulfation reactions occur post-translationally on numerous extracellular matrix macromolecules prior to secretion and mineralization. The Na(+)/H(+)exchanger proteins are regulated by mitogenic and non-mitogenic stimuli in the osseus environment and are involved in the large fluxes of ions and protons that necessarily occur during bone formation and resorption and thus play an important role in intracellular ion homeostasis in osteoblasts.