Protein kinase a-dependent spinal α-amino-3-hydroxy-5-methyl-4- isoxazoleproprionate-receptor trafficking mediates capsaicin-induced colon-urethra cross-organ reflex sensitization

Background: Intracellular redistribution of α-amino-3-hydroxy-5- methyl-4-isoxazoleproprionate receptors (AMPARs) is known to be induced by natural painful stimulation. We tested the hypothesis that that protein kinase A (PKA)-dependent AMPAR trafficking underlies the development of N-methyl-d-aspartate receptor-mediated cross-organ sensitization in vivo. Methods: We recorded urethra reflex activity and analyzed immunoblotting of lumbosacral (L6-S2) dorsal horn (DH) tissue obtained from animal preparations after intrathecal 8-bromo-cyclic adenosine monophosphate injection or intracolonic instillation with 8-methyl-N-vanillyl-trans-6-nonenamide (capsaicin). Results: Intrathecal 8-bromo-cyclic adenosine monophosphate (300 μM, 10 μl) induced reflex potentiation (81.85 ± 22.21 spikes/stimulation) and increased the number of AMPAR Glu receptor 1 subunits in the membrane fraction of DH (1.8-fold increase vs. control). This process was prevented by pretreatment with the PKA inhibitor N-[2- ((p-bromocinnamyl)amino) ethyl]-5-isoquinolinesulfonamide(10 μM, 10 μl, 2.7 ± 0.8 [mean ± SE] spikes/stimulation) and human thyroid A kinase-anchoring protein (10 μM, 10 μl, 11.5 ± 4.8 spikes/stimulation), an inhibitor of PKA and PKA-A kinase-anchoring protein interactions. Intracolonic capsaicin instillation sensitized the urethra reflex (137.2 ± 62.4 spikes/stimulation) and, relative to control, simultaneously provoked an increase (2.9-fold) in the membrane fraction and a decrease (0.9-fold) in the cytosolic fraction of Glu receptor 1 subunits in DH. Inhibition of PKA activity and disruption of PKA-A kinase-anchoring protein interaction in the DH (2.0 ± 0.6 and 16.7 ± 2.8 spikes/stimulation, respectively) are sufficient to prevent capsaicin-dependent reflex sensitization and AMPAR trafficking in the membrane fraction (0.6- and 0.5-fold increase capsaicin). Conclusion: Delivery of AMPAR-containing Glu receptor 1 subunits to the membranes of lumbosacral DH neurons through a PKA-dependent pathway contributes to noxious stimulation-induced synaptic strengthening, which plays roles in colon-urethra reflex sensitization.