NMDA receptor expression and activity in osteoarthritic human articular chondrocytes.
OBJECTIVE
Classical neuronal signalling molecules such as substance P and glutamate have been identified in cartilage and have roles in regulation of chondrocyte function. This study looks at expression and activity of the ionotropic glutamate NMDA (N-methyl-D-aspartic acid) receptor (NMDAR) in human osteoarthritic (OA) chondrocytes.


METHODS
Chondrocytes were obtained from human knee joint arthroplasty specimens. NMDAR subunits and PSD-95 (postsynaptic density protein 95) expression were analysed by reverse transcription-polymerase chain reaction and Western blotting. Activity of NMDAR was assayed by radioactive calcium(45) uptake and changes in membrane potential in the presence and absence of NMDA and NMDAR antagonists and blockade of cell membrane ion channels.


RESULTS
NMDAR 1, 2A, 2B and PSD-95 were detected in human OA chondrocytes whereas NR2B was absent from normal chondrocytes. NMDA induced calcium flux into OA chondrocytes and cell membrane depolarisation. These responses were blocked by NMDAR antagonists, removal of extracellular calcium, inhibition of nNOS (neuronal nitric oxide synthase) activity and uncoupling of NMDAR from PSD-95. Blockade of sodium channels by tetrodotoxin resulted in NMDA-induced membrane hyperpolarisation which was, in turn inhibited by apamin, a blocker of SK channels. NMDA-induced changes in cell membrane potential were not affected by l-type and stretch activated calcium channel inhibitors.


CONCLUSIONS
Human OA and normal articular chondrocytes differ in the expression of NMDAR subunits. In OA chondrocytes NMDAR signalling requires extracellular calcium, association with PSD-95, and nNOS activity. Downstream signalling results in activation of tetrodotoxin sensitive sodium channels and SK channels, a response that differs from that of normal chondrocytes suggesting altered activity of NMDAR in OA.
Centre for Inflammation Research, The Queens Medical Research Institute, The University of Edinburgh, Edinburgh, UK. lindsayramage@hotmail.co.uk
L
L
Ramage
M-A
MA
Martel
G E
GE
Hardingham
D M
DM
Salter
D015220:Calcium Channels / Q000378:metabolism*; D002358:Cartilage, Articular / Q000166:cytology / Q000378:metabolism*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D020370:Osteoarthritis, Knee / Q000235:genetics / Q000378:metabolism*; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000235:genetics; D013582:Synovial Fluid / Q000166:cytology / Q000378:metabolism*
D015220:Calcium Channels; D016194:Receptors, N-Methyl-D-Aspartate