In the lumbar spinal cord dorsal horn, release of afferent nerve
glutamate activates the neurons that relay information about injury
pain. Here, we examined the effects of
protein tyrosine kinase (PTK) inhibition on
NMDA receptor NR1 subunit protein expression and subcellular localization in an acute
experimental arthritis model. PTK inhibitors
genistein and
lavendustin A reduced cellular histological translocation of
NMDA NR1 in the spinal cord occurring after the inflammatory insult and the nociceptive behavioral responses to heat. The PTK inhibitors were administered into lumbar spinal cord by microdialysis, and secondary heat
hyperalgesia was determined using the Hargreaves test.
NMDA NR1 cellular
protein expression and nuclear translocation were determined by immunocytochemical localization with light and electron microscopy, as well as with Western blot analysis utilizing both C- and N-terminal
antibodies.
Genistein and
lavendustin A (but not inactive
lavendustin B or
diadzein) effectively reduced (i)
pain related behavior, (ii)
NMDA NR1 subunit expression increases in spinal cord, and (iii) the shift of NR1 from a cell membrane to a nuclear localization.
Genistein pre-treatment reduced these events that occur in vivo within 4 h after inflammatory insult to the knee joint with
kaolin and
carrageenan (k/c).
Cycloheximide reduced
glutamate activated upregulation of NR1 content confirming synthesis of new
protein in response to the inflammatory insult. In addition to this in vivo data,
genistein or staurosporin inhibited upregulation of
NMDA NR1
protein and nuclear translocation in vitro
after treatment of human
neuroblastoma clonal cell cultures (SH-SY5Y) with
glutamate or
NMDA (4 h). These studies provide evidence that inflammatory activation of peripheral nerves initiates increase in
NMDA NR1 in the spinal cord coincident with development of
pain related behaviors through
glutamate non-
receptor, PTK dependent cascades.