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*Department of Anesthesiology, Kyoto Prefectural University of Medicine; and Graduate School of Pharmaceutical Sciences, Kyoto University, Japan
Address correspondence and reprint requests to Munetaka Hirose, MD, Department of Anesthesiology, Kyoto Prefectural University of Medicine, Kamigyoku, Kyoto 602-8566, Japan. Address e-mail to hirose{at}koto.kpu-m.ac.jp.
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Abstract |
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Introduction |
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TrkA, a high affinity receptor of NGF, is a receptor tyrosine kinase (RTK) appearing early in the embryonic development of dorsal root and trigeminal neurons and later, near the time of birth, in sympathetic neurons (4). NGF binding to the extracellular portion of TrkA leads to autophosphorylation of 5 different tyrosine residues, located in the juxtamembrane domain (Y490), in the activation loop of the tyrosine kinase domain (Y670, Y674, and Y675), and in the C-terminal (Y785) (5,6). The amino acid sequences of activation loop including these three tyrosine residues (Y670, Y674, and Y675) in TrkA reveal homology with a number of other RTKs, e.g., the insulin receptor, the insulin-like growth factor receptor, the receptors for brain-derived neurotrophic factor (BDNF) (TrkB), and neurotrophin-3 (TrkC), as shown in Table 1, and are important for initial ligand-induced autophosphorylation (7,8). NGF-stimulated autophosphorylation of three tyrosines in the activation loop of TrkA regulates both overall and specific downstream signaling and promotes biological responses, including neurite outgrowth (5).
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Our previous studies revealed that the LA lidocaine directly inhibits ligand-stimulated autophosphorylation of RTKs, i.e., the insulin receptor and the epidermal growth factor receptor, and suppresses downstream signaling through binding with the autophosphorylation sites (9–11). Because of a close resemblance of the amino acid sequences in the activation loop between the insulin receptor and TrkA (Table 1), we hypothesized that inhibition of NGF-stimulated tyrosine kinase activity of TrkA would be a plausible mechanism of suppression of neurite outgrowth by LAs. In the present study, we investigated the effect of LAs on NGF signaling using a PC12 cell line derived from rat pheochromocytoma, which is a useful model for studying NGF signaling and neurite outgrowth (12,13).
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Methods |
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PC12 cells were purchased from American Type Culture Collection (Manassas, Virginia) and maintained on 100-mm diameter culture dishes at 37°C under an atmosphere of 5% CO2 and 95% air. Cells were grown in RPMI 1640 medium supplemented with 5% fetal bovine serum, 10% horse serum, 2 mM of l-glutamine, penicillin, and streptomycin. The culture medium was changed twice per week.
Cells were grown in 24-well dishes with or without NGF (100 ng/mL) and LA (lidocaine, bupivacaine, or procaine) for an indicated time, and the outgrowth of neurites was monitored using phase-contrast microscopy. Processes with lengths equivalent to one or more diameters of a cell body were counted as neurites. A minimum of 100 cells was examined for each datum point.
To analyze the effect of these LAs on NGF-stimulated autophosphorylation of TrkA, the PC12 cells were cultured on 100-mm-diameter dishes to be 80% confluent. NGF (100 ng/mL) or LA (40 µM, 400 µM, and 4000 µM) were added, and then cells were incubated in a 37°C 5% CO2 environment for 5 min. Each medium was replaced with 5 mL of ice cold phosphate-buffered saline, and each sample was scraped and harvested into the tubes. The supernatant was removed by centrifugation at 1500 rpm for 5 min. Each sample was suspended in 0.6 mL of lysis buffer (HEPES, with a pH value of 7.5, 50 mM, NaCl 150 mM, EDTA 2 mM, 1% (vol/vol) Nonidet P-40, 10% (vol/vol) glycerol, sodium fluoride 10 mM, sodium vanadate 2 mM, phenylmethylsulphonyl fluoride 1 mM, sodium pyrophosphate 10 mM, 5 µg/mL of aprotinin, and 0.5 µg/mL of pepstatin) and laid on ice for 30 min. Insoluble material was removed by centrifugation at 15,000 rpm for 15 min. Aliquots of the supernatants containing equal amounts of protein, as determined using the Bradford protein assay with Bradford reagent, were subjected to immunoprecipitation for 1 h at 4°C with anti-Trk antibody. After the addition of protein A-Sepharose CL-4B, the immunoprecipitates were washed 3 times in a wash buffer (HEPES, with a pH value of 7.5, 50 mM, NaCl 150 mM, EDTA 2 mM, 0.1% (vol/vol) Nonidet P-40, 10% (vol/vol) glycerol, sodium fluoride 10 mM, sodium vanadate 2 mM, phenylmethylsulphonyl fluoride 1 mM, sodium pyrophosphate 10 mM, 5 µg/mL of aprotinin, and 0.5 µg/mL of pepstatin). Each sample was prepared for sodium dodecyl sulfate-polyacrylamide gel electrophoresis by adding Laemmli sample buffer and boiling for 5 min. The immunoprecipitate was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 7.5% (vol/vol) acrylamide solving gels and transferred electrophoretically to nitrocellulose membrane. The membrane was then blocked in 5% (wt/vol) dried milk in phosphate-buffered saline containing 0.1% (vol/vol) polyoxymethylene sorbitan monolaurate (Tween 20) for 1 h at room temperature and was then immunoblotted with the appropriate antibody. Although anti-Trk antibodies detect all three Trks (TrkA, TrkB, and TrkC), tyrosine phosphorylated protein, stimulated by NGF to approximately a 140-kDa level, can be identified as autophosphorylated TrkA because NGF binds to only TrkA. The antigen antibody complexes were visualized by a chemiluminescence luminol reagent. Bands of interest were scanned and quantified by using LightCapture AE-6960 (ATTO Corporation, Tokyo, Japan).
After incubation with NGF, LA, or both for 3 days, cells were stained with Hoechst 33342 (1 µg/mL) and propidium iodide (PI) (1 µg/mL) for 10 min and analyzed under a fluorescence microscope. Because Hoechst 33342 (blue) stains all nuclei and PI (red) stains nuclei of cells with a disrupted plasma membrane, dead cells were observed as red nuclei. Cell death was also quantified by measuring lactate dehydrogenase (LDH) released from dead cells after 3 days of incubation with NGF, LA, or both using the LDH-Cytotoxic Test (Wako, Osaka, Japan).
Data were analyzed by one-way analysis of variance with Scheffe post hoc analysis. The statistical significance was established at the P < 0.05 level. All values are reported as mean ± sd.
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Results |
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Figure 2 shows time course of NGF-stimulated neurite outgrowth of PC12 cells under exposure of lidocaine (0, 40, 400, 4000 µM) for 1, 3, and 4 days. Lidocaine (400 µM) significantly suppressed neurite outgrowth on every 1, 3, and 4 day compared to that without lidocaine, and 4000 µM of lidocaine did not induce neurite outgrowth throughout the study.
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To assess tyrosine phosphorylation of TrkA, cell lysis was subjected to immunoprecipitation with the anti-Trk antibody followed by immunoblotting with the antiphosphotyrosine antibody. NGF stimulation resulted in a marked increase in tyrosine phosphorylation of TrkA in PC12 cells. Lidocaine (400 µM and 4000 µM), bupivacaine (40 µM, 400 µM, and 4000 µM), or procaine (4000 µM) significantly attenuated the NGF-stimulated tyrosine phosphorylation of TrkA in the PC12 cells relative to control (100%) (Fig. 3). Protein levels did not differ among these groups.
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Cytotoxicity of these LAs was assayed by LDH release and by the morphological features of the cells using double staining with Hoechst 33342 and PI under fluorescence microscope. Hoechst 33342 (blue) stains all nuclei, and PI (red) stains only nuclei in cells with disrupted membrane integrity. PC12 cells after exposure to 4000 µM of lidocaine or bupivacaine for 3 days showed significantly increased PI positive cells (Fig. 4). The LDH assay also showed that only 4000 µM of lidocaine or bupivacaine significantly increased cell death (Fig. 4).
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Taken together, 4000 µM of lidocaine or bupivacaine had both effects of cytotoxicity and neurite outgrowth reduction in PC12 cells. However, lidocaine (400 µM), bupivacaine (40 and 400 µM), or procaine (4000 µM) suppressed neurite outgrowth after the suppression of NGF-stimulated autophosphorylation of TrkA, without a cytotoxic effect on PC12 cells.
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Discussion |
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- and C-fibers in rat sciatic nerve (15). Therefore, 400 µM of lidocaine corresponds to the concentration of around or in the peripheral nerve for local or regional anesthesia. The smallest concentration of lidocaine (40 µM) in the present study, however, was somewhat larger than the plasma concentration after IV lidocaine injection for neuropathic pain (16,17). The present results showed that lidocaine only suppressed NGF-stimulated TrkA tyrosine kinase activity and neurite outgrowth at the concentrations of 400 µM or larger. Bupivacaine (40 µM or larger) or procaine (4000 µM) also suppressed them. The potential order of the inhibition of NGF-stimulated TrkA is bupivacaine > lidocaine > procaine, as shown in the present results. However, a recent in vivo study reported that when using animal models of neuropathic pain, both continuous nerve block (2% lidocaine) and systemic administrations of lidocaine (plasma lidocaine level ranged between 0.5 and 1.4 µg/mL, equivalent to between 2 and 6 µM) decreased nerve injury-induced sympathetic sprouting in the dorsal root ganglia, which is a major phenomenon implicated in neuropathic pain (1). In the in vitro study using PC12 cells, 10 µg/mL (approximately 30 µM) of ropivacaine showed no effect on NGF-mediated neurite outgrowth, and larger concentrations (more than 300 µM) of ropivacaine inhibited it (3). Although it is difficult to compare the results between in vivo and in vitro studies, we suggest that mechanisms of suppression of sympathetic sprouting by LA or regional anesthesia likely involve the inhibitory effect of LAs on NGF-stimulated tyrosine kinase activity of TrkA.
Our previous study revealed that there are interactions between lidocaine and the activation loop of insulin receptor, including aromatic amino acids (Y1158, Y1162, and Y1163), basic amino acids (R1155 and R1164), and acidic amino acids (D1156, E1159, and D1161), with a variety of combinations of noncovalent interactions (9,10). Because of a close resemblance of the amino acid sequences in the activation loop between the insulin receptor and TrkA (Table 1), it can be considered that lidocaine binds to tyrosine residues and acidic and basic amino acids in the activation loop of TrkA. The molecular interactions between LAs and the activation loop of TrkA would be a mechanism of the inhibition of TrkA activity by LAs. The weakness of the present study, however, includes the lack of the investigation using purified TrkA in vitro. Further study is required to investigate the direct effect of LAs on TrkA activity using purified TrkA.
Although the precise mechanism of the inhibitory effect of LAs on NGF-mediated neurite outgrowth has not been evaluated, other investigators reported that cocaine inhibits neurite outgrowth through D1-type dopamine receptors (18). They proposed that cocaine binds to dopamine transporters, resulting in suppression of dopamine reuptake and increased dopamine level in nerve endings, and then dopamine directly inhibits NGF-mediated neurite outgrowth of PC12 cells. Other LAs, however, have only a partial inhibitory effect of dopamine reuptake (19). Therefore, several mechanisms, including the TrkA inhibition and the increased dopamine reuptake, seem to play a role in the suppression of neurite outgrowth by LAs.
A large concentration of LAs has a cytotoxic effect on neuronal cells with activation of apoptotic pathways. A dorsal root ganglion cell line shows cell death after 24-hour incubation with 2300 µM of lidocaine (20). Another study reported that incubation with 1000 µM of lidocaine, bupivacaine, or procaine for 10 hours does not affect cell survival in PC12 cells, and 2000 µM of these LAs, except procaine, induce cell death (21). Both 4000 µM of lidocaine and bupivacaine suppressed either NGF-mediated neurite outgrowth or NGF-stimulated autophosphorylation of TrkA with cytotoxicity in the present study. In addition to the inhibitory effect of these LAs on TrkA activity, the cytotoxic effect in these large concentrations of lidocaine and bupivacaine is also involved in the mechanisms of complete reduction of neurite outgrowth. Procaine, which is less toxic to neurons than lidocaine and bupivacaine, suppressed NGF-mediated neurite outgrowth without cytotoxicity in this large concentration.
Epidural injection of LAs periodically or continuously is used to reduce the incidence of postherpetic neuralgia, which causes neuropathic pain after herpes zoster (22). In addition to the important factor for sympathetic sprouting, NGF, which is released by mast cells, fibroblasts, and other cell types at sites of injury and inflammation, is also important for nociception and long-lasting pain in conditions of inflammation and induces peripheral sensitization in the primary sensory neurons and central sensitization in the spinal dorsal horn neurons through activation of extracellular signal-regulated protein kinase with an increase of substance P and BDNF expressions (23,24). Studies indicate that BDNF synthesized in the dorsal root ganglion cells activates TrkB in the spinal dorsal horn neurons, and that is involved in some aspects of central sensitization in conditions of peripheral inflammation (25). Because there is a homology of amino acid sequences among Trks, i.e., TrkA, TrkB, and TrkC (Table 1), it is likely that Trks might be important targets of LAs for antinociception in the conditions of peripheral inflammation by suppression of both peripheral and central sensitization.
In summary, the LAs at the concentration around or in the peripheral nerve after local administration suppressed NGF-stimulated autophosphorylation of TrkA in PC12 cells with the reduction of neurite outgrowth. The inhibition of TrkA activity might be involved in the mechanisms of the suppression of neurite outgrowth by LAs.
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Footnotes |
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Accepted for publication September 13, 2005.
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This article has been cited by other articles:
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  H. Hiruma, K. Shimizu, T. Takenami, H. Sugie, and T. Kawakami Effects of clonidine on lidocaine-induced inhibition of axonal transport in cultured mouse dorsal root ganglion neurones Br. J. Anaesth., November 1, 2008; 101(5): 659 - 665. [Abstract] [Full Text] [PDF]
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 C. Sato, A. Sakai, Y. Ikeda, H. Suzuki, and A. Sakamoto The Prolonged Analgesic Effect of Epidural Ropivacaine in a Rat Model of Neuropathic Pain Anesth. Analg., January 1, 2008; 106(1): 313 - 320. [Abstract] [Full Text] [PDF]
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