Cells of mammalian bone express glutamate receptors. Functional N-methyl-D-aspartate (NMDA) receptors have been demonstrated in human, osteoblastic MG-63 cells, but currents in these cells, unlike those of mammalian neurons, are blocked by Mg(2+) in a voltage-insensitive manner. Differences between the characteristics of NMDA currents in bone cells and in neurons may reflect molecular variation of the receptors or associated molecules, with implications for the role(s) of glutamate in these different tissues and for targeting of ligands/antagonists. To determine whether NMDA receptors in primary bone cells are functional, and whether the currents carried by these receptors resemble those of MG-63 cells or those of mammalian neurons, we have applied the whole cell patch clamp technique to primary cultures of rat osteoblasts. In 0-Mg(2+) saline, 25% of cells showed a slowly developing inward current in response to bath perfusion with 1 mM or 100 microM NMDA. Antibodies against NMDA receptors stained approximately 26% of cells. When NMDA was applied by rapid superfusion, kinetics of the currents were similar to those of neuronal NMDA currents, reaching a peak within 20-30 ms. 1 mM Mg(2+) reduced current amplitude at negative holding potentials and caused the I-V relationship of the currents to adopt a 'J' shape rather than the linear relationship seen in the absence of added Mg(2+). Co-application of glycine (20 microM) with NMDA increased current amplitude by only 18%, suggesting that glycine is released from cells within the cultures. Currents were blocked by (+)-MK-801 and DL-2-amino-5-phosphonovaleric acid. Fluorimetric monitoring of [Ca(2+)](i) using fura-2 showed that, in Mg(2+)-free medium, NMDA caused a sustained rise in [Ca(2+)](i) that could be reversed by subsequent application of MK-801. We conclude that rat femoral osteoblasts express functional NMDA receptors and that these receptors differ from those previously identified in MG-63 cells. NMDA receptors of primary osteoblasts show a 'classical' voltage-sensitive Mg(2+) block, similar to that seen in neuronal NMDA receptors, and will therefore function as detectors of coincident receptor activation and membrane depolarization.
- Dizocilpine Maleate
- Excitatory Amino Acid Antagonists
- Membrane Potentials
- Patch-Clamp Techniques
- Receptors, N-Methyl-D-Aspartate