Molecular identification of NMDA glutamate receptors expressed in bone cells

The N-methyl-D-aspartate (NMDA) subtype of the glutamate receptor has recen tly been identified in bone, but the molecular composition of this receptor expressed by bone cells is unknown. NMDA receptor (NMDAR) is a hetero-olig omeric protein composed of two classes of subunits, the essential subunit N R1 and NR2A to D subunits that do not by themselves produce functional chan nels but potentiate NR1 activity and confer functional variability to the r eceptor. These subunits coassemble in different combinations to form functi onally distinct NMDAR. In this study, we have investigated the molecular co mposition of NMDAR expressed by osteoblasts and osteoclasts in culture, usi ng RT-PCR analysis, in situ hybridization and immunocytochemistry. Specific probes were designed for the different subunits of the NMDAR, and we showe d by RT-PCR analysis that mammalian osteoclasts expressed NR2B and NR2D sub units mRNAs but not NR2A and NR2C mRNAs. Rat calvaria and MG63 osteoblastic cells also expressed several NR2 subunits mRNAs, namely NR2A, NR2B, and NR 2D. In situ hybridization on isolated rabbit osteoclasts and MG63 cells has confirmed the localization of NR1, NR2B, and NR2D transcripts in osteoclas ts: and NR1, NR2A, NR2B, and NR2D transcripts in MG63 cells. The expression of NR2D protein by bone cells was shown by immunofluorescence. These resul ts demonstrate for the first time that osteoblasts and osteoclasts express several NR2 subunits, suggesting a molecular diversity of NMDAR channels si milar to what was shown for brain. The presence of distinct functional NMDA R on bone cells may be associated with various states of bone cell differen tiation and function.