Objective: 

N-methyl-D-aspartate type glutamate receptors (NMDARs) are non-selective cationic channels with high Ca²⁺-permeability and require glycine coagonist to become activated. Functional NMDAR is a heterotetramer and NR1 subunit is essential to the receptor assembly and trafficking to the plasmamembrane. The presence of glycine-binding NR3 subunit decreases the Ca²⁺-permeabilty. Previously, we had reported characteristic changes in the basal cytosolic Ca²⁺-concentration [Ca²⁺]I in chondrogenic cells of chondrifying high density cell cultures (HDC). Here we investigated the role of the NMDAR in chondrogenesis.

Methods: 

We detected expression of NR1, NR2A, NR2B, NR3A and NR3B subunits in HDC, but only NR1, NR2B and NR3B were present on Western blots of plasma-membrane fraction, with the highest expression on day 2 of culturing, when final commitment of chondrogenic cells occurs.

Results: 

We recorded rapid spontaneous Ca²⁺ -oscillations responding to NMDA with longer duration. NMDA also generated a pronounced elevation of basal [Ca²⁺]i. Pre-incubation with ifenprodil (an NR2B-specific NMDAR inhibitor) suppressed the calcium transients. On whole cell patch clamp records weak inward current became visible in the presence of NMDA with NMDG based internal solution. These responses to NMDA were the most pronounced on day 2. The agonist glutamate and NMDA both failed to influence cartilage formation, but both glycine and ifenprodil promoted chondrogenesis. Transient gene silencing via introduction of NR1 siRNA, blocked cartilage formation and almost completely abolished spontaneous Ca²⁺ oscillations.

Conclusions: 

Our results prove the profound role of NMDARs in the regulation of chondrogenesis via influencing [Ca²⁺]i of differentiating cells.