Local anesthetics (LAs) suppress sympathetic sprouting, which correlates with neuropathic pain. However, the precise mechanism of the suppression is unknown. Nerve growth factor (NGF) contributes to the sympathetic sprouting, and NGF signaling starts with NGF-stimulated autophosphorylation of TrkA, which is a high affinity receptor of NGF. We examined the effects of lidocaine, bupivacaine, and procaine on NGF signaling under suppression of NGF-stimulated neurite outgrowth in PC12 cells, which is a cellular model of sympathetic sprouting. To investigate the effect of these LAs on NGF-mediated neurite outgrowth of PC12 cells, cells were incubated with 40, 400, and 4000 microM of each LA. The effect of LAs on NGF-stimulated TrkA activity was examined to analyze autophosphorylation of TrkA using immunoprecipitation and immunoblotting. Cytotoxic effects of LAs on PC12 cells were also assessed by lactate dehydrogenase release and by propidium iodide staining. Lidocaine (400 microM), bupivacaine (40 and 400 microM), or procaine (4000 microM) suppressed either neurite outgrowth or autophosphorylation significantly without cytotoxicity. The inhibition of NGF-stimulated tyrosine kinase activity of TrkA might be involved in the mechanisms of suppression of neurite outgrowth induced by LAs.

Abstract. As TrkA, a high-affinity receptor of nerve growth factor (NGF), is a potential target for relieving uncontrolled inflammatory pain, an effective inhibitor of TrkA has been required for pain management. To identify a specific inhibitor of TrkA activity, we designed cell-penetrating peptides combined with amino-acid sequences in the activation loop of TrkA to antagonize tyrosine kinase activity. To select a peptide inhibiting TrkA activity, we examined the effect of cell-penetrating peptides on tyrosine kinase activity of recombinant TrkA in vitro and studied their effects on NGF-stimulated neurite outgrowth and protein phosphorylation in PC12 cells. Thereafter we investigated the effect of the selected peptide on NGF-stimulated TrkA activity and the expression of transient receptor potential channel 1 in PC12 cells. The selected peptide inhibited TrkA activity, but did not inhibit tyrosine kinase activities of other receptor-type tyrosine kinases in vitro. It also suppressed NGF-stimulated responses in PC12 cells. The selected synthetic cell-penetrating peptide antagonizing TrkA function would be a candidate for inflammatory pain therapy.

Abstract. Novel agents that inhibit nerve growth factor signaling are required for the treatment of inflammatory pain. The present study investigated the effect of local administration of inhibitory peptide of TrkA (IPTRK3), a synthetic cell-penetrating peptide that antagonizes TrkA function, in complete Freund's adjuvant (CFA)-induced hyperalgesia in rats. Three hours after subcutaneous injection of CFA into the plantar surface of the rat's left hind paw, 10 mM IPTRK3 was injected at the same site. Thermal and mechanical hyperalgesia were tested in the ipsilateral hind paw until 7 days after CFA injection. The ipsilateral dorsal root ganglion (DRG) was dissected out for immunohistochemical analysis of transient receptor potential vanilloid subfamily member 1 (TRPV1) channels and TrkA. Local injection of this peptide significantly suppressed both thermal and mechanical hyperalgesia produced by CFA and also significantly reduced TRPV1 expression at the DRG. These results suggest that local administration of IPTRK3 is likely effective in the treatment of inflammatory pain in rats.

A unilateral deep thoracic wound produces pain-related behavior and, at the same time, ipsilateral upregulation of microglial COX-1 and neuronal COX-2 in the thoracic dorsal horn.

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