Abstract
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.
Highlights
A critical and unresolved issue in activation dependent neuronal signaling is identification of the numerous signaling molecules and matching them with their specific roles
As we have reported previously in this inflammatory arthritis model in rats, reduced paw withdrawal latency (PWL) is indicative of central sensitization and secondary heat hyperalgesia (Sluka and Westlund, 1993a)
The studies with the protein tyrosine kinase (PTK) inhibitors provide evidence that protein tyrosine phosphorylation is important in (i) modulation of nociceptive responses induced by peripheral k/c arthritis, (ii) induced NMDA NMDA receptor 1 (NR1) subunit expression increases in the spinal cord, and (iii) NMDA NR1 subunit trafficking from the cell membrane to the nuclear membrane
Summary
A critical and unresolved issue in activation dependent neuronal signaling is identification of the numerous signaling molecules and matching them with their specific roles. Non-receptor Src family protein tyrosine kinase (PTK) interactions with tyrosine residues of ionotropic NMDA receptor NR2A/B subunits impact phosphorylation, channel protein insertion, and activation state, in the hippocampus as it impacts long term potentiation (LTP). NR1 expression increase in the lumbar spinal cord was initiated by knee joint inflammation with k/c injection. Two PTK inhibitors (genistein and lavendustin A) and their inactive analogs (diadzein and lavendustin B) were investigated to determine their influence on pain related behavior, spinal cord content and cellular localization of NR1 after inflammatory hindlimb insult. The PTK inhibitors reduced spinal cord NR1 neuronal expression and nuclear translocation in the spinal cord, as well as improved secondary heat hypersensitivity on the footpad in an experimental knee joint arthritis
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