Activation of protein kinase B/Akt signaling pathway... : PAIN (original) (raw)
1. Introduction
The mechanisms associated with chronic pain states are not clearly understood. However, a growing body of evidence indicates that the synaptic plasticity of dorsal horn neurons contributes to pain hypersensitivity (central sensitization) after strong noxious stimulation (Guo et al., 2002, 2004; Ji et al., 2003; Willis, 2002; Woolf and Salter, 2000). Central sensitization is thought to underlie the development of secondary hyperalgesia and allodynia following tissue injury (Simone et al., 1989; Sun et al., 2004b; Torebjork et al., 1992; Willis, 2002). It is thought that central sensitization is triggered by glutamate acting on _N_-methyl-d-aspartate (NMDA), non-NMDA and group I metabotropic receptors; SP acting on NK-1 receptors; CGRP acting on CGRP1 receptors; and BDNF acting on tyrosine kinase B receptors (TrkB) (Dougherty et al., 1994, 1995; Garraway et al., 2003; Karim et al., 2001; Mannion et al., 1999; Neugebauer et al., 1999, 2000; Pezet et al., 2002a,b; South et al., 2003; Sun et al., 2003, 2004a,b,c; Thompson et al., 1999). Several signaling pathways mediate the effects of these receptors in nociceptive dorsal horn neurons that participate in central sensitization, such as the protein kinase A (Lin et al., 2002; Zou et al., 2002) protein kinase C (Guo et al., 2002, 2004; Lin et al., 1996; Palecek et al., 1994, 1999; Sluka et al., 1997; Zou et al., 2004), NO/protein kinase G (Lin et al., 1997, 1999a,b; Wu et al., 1998, 2001), calcium/calmodulin-dependent protein kinase II (Fang et al., 2002) and extracellular signal-regulated kinase (ERK) cascades (Adwanikar et al., 2004; Ji et al., 1999; Karim et al., 2001; Kawasaki et al., 2004).
Protein kinase B/Akt (PKB/Akt) is a critical downstream target of phosphoinositide 3-kinase (PI3K) and mediates the key functions of the PI3K dependent survival pathway through its phosphorylation and regulation of apoptotic proteins and transcription factors (Ashcroft et al., 1999; Barber et al., 2001; Brunet et al., 2001; Dudek et al., 1997; Dolcet et al., 1999; Rodgers and Theibert, 2002). PKB/Akt is activated by phosphorylation on Thr308 and Ser473 (Alessi and Cohen, 1998; Alessi et al., 1996; Meier et al., 1997; Toker and Newton, 2000). Recent studies indicate that the PI3K-PKB/Akt signaling pathway is involved in synaptic plasticity in the brain (Hou and Klann, 2004; Izzo et al., 2002; Kelly and Lynch, 2000; Lin et al., 2001; Opazo et al., 2003; Sanna et al., 2002). The phosphorylation of PKB/Akt is significantly increased during long-term potentiation (LTP) and long-term depression (LTD).
Current evidence supports the view that synaptic plasticity in the hippocampus and central sensitization in the spinal cord share common signaling pathways (Fang et al., 2002; Ji et al., 1999; Kawasaki et al., 2004; Willis, 1997, 2002). We hypothesize that activation of the PKB/Akt pathway contributes to the mechanical hypersensitivity induced by capsaicin. We used immunohistochemistry, Western blots, and behavioral testing experiments to study whether the PKB/Akt pathway is involved in pain hypersensitivity. Preliminary findings have been reported (Sun et al., 2004a,b,c).
2. Materials and methods
2.1. Animals and materials
Male Sprague–Dawley rats weighing 250–320g were used in this study. The experiments were approved by the Institutional Animal Care and Use Committee and were consistent with the ethical guidelines of the National Institutes of Health and of the International Association for the Study of Pain. All experimental animals were housed and maintained in accordance with the guidelines of the University of Texas Medical Branch Animal Care and Use Committee.
Phospho-Akt antibody (473) was purchased from Cell Signaling Technology (Beverly, MA). β-actin monoclonal antibody was purchased from Sigma (St Louis, MO). Neuronal nuclei (NeuN) monoclonal antibody was purchased from Chemicon (Temecula, CA). The horseradish peroxidase-linked goat anti-rabbit IgG and goat anti-mouse IgG were obtained from BIO-RAD (Hercules, CA). Alexa Fluor® 488 goat anti-mouse secondary antibody and Alexa Fluor® 594 goat anti-rabbit secondary antibody were obtained from Molecular Probes (Eugene, OR). Enhanced chemiluminescence (ECL) Western blotting detection reagents were obtained from Amersham Biosciences (Piscataway, NJ). All PI3K and PKB/Akt inhibitors were purchased from Calbiochem (La Jolla, CA).
2.2. Western immunoblotting
Spinal cord tissue (L3–L5) was collected. The dorsal quadrants were homogenized in ice-cold homogenization buffer (HB) containing phosphatase and protease inhibitors (200nM calyculin, 10μg/ml leupeptin, 2μg/ml aprotinin, 1mM sodium orthovanadate, and 1μM microcystin-LR).The homogenate was centrifuged at 13,000×g for 15min at 4°C. The supernatant for the cytosolic fraction was decanted from the pellet and used for all Western blot analyses. Equivalent amounts of protein for each sample were dissolved in 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were blocked in 5% nonfat dry milk for 1h in Tris-buffered saline containing Tween 20 and then incubated with the phospho-Akt (Ser473) antibody overnight at 4°C followed by incubation with horseradish peroxidase-linked goat anti-rabbit IgG (1:2500 dilution) and developed using ECL. The blots then were incubated in stripping buffer (62mM Tris–HCl, pH 6.8, 2% SDS, and 100 mM β-mercaptoethanol) for 1h at 55–60°C followed by incubation in Tris-buffered saline with Tween 20 for 30min. The stripped blots were incubated with β-actin antibody (1:25,000). Densitometric analysis of phospho-Akt (pAkt) immunoreactivity and β-actin immunoreactivity was conducted using Metamorph offline software. β-actin immunoreactivity was used as a loading control. A single band for pAKT in Western blots confirmed the specificity of the antibody. One way ANOVA followed by a Newman-Keuls test was performed to compare groups.
2.3. Immunohistochemistry
Five minutes after intradermal capsaicin injection, animals were deeply anesthetized with sodium pentobarbital (50mg/kg, i.p) and perfused through the ascending aorta with warm heparinized saline, followed by 4% paraformaldehyde (4°C). After the perfusion, the lumbar segments of the spinal cord were removed and placed in fresh fixative for 4h. The fixative was then replaced with 30% sucrose overnight. Spinal cord sections (14μM) were cut in a cryostat and processed for immunofluorescence. All of the sections were blocked with 5% normal goat serum in PBS/0.3% Triton X-100 (PBS-TX) for 2h at room temperature. Sections were then incubated overnight at 4°C with primary antibodies [phospho-Akt antibody (ser 473)]. After washing three times with PBS-TX, sections were incubated for 2h at room temperature (RT) with Alexa Fluor® 594 goat anti-rabbit IgG. For double immunofluorescence, sections were incubated with a mixture of phospho-Akt antibody (ser 473) and monoclonal neuronal-specific nuclear protein (NeuN) antibody (1:500) overnight at 4°C, followed by a mixture of Alexa Fluor® 594 goat anti-rabbit IgG and Alexa Fluor® 488 goat anti-mouse secondary antibodies for 2h at room temperature. To confirm the specificity of the immunolabeling, control slides were exposed to dilute normal goat serum instead of the primary antibody. Control slides that omitted the primary antibody were consistently negative. The specificity of the antibody has also been tested in previous studies (Zhuang et al., 2004).
2.4. The placement of intrathecal catheters
Externally accessible PE-10 intrathecal (i.t.) catheters were implanted in rats to be used for behavioral testing, according to a modification of the technique described by Storkson et al. (1996). After anesthesia with sodium pentobarbital (50mg/kg. i.p.), a catheter (PE 10, o.d. 0.61mm) was inserted into the subarachnoid space through a guide cannula connected to a 20-gauge needle, which punctured the dura at the level of the cauda equina. The catheter was then carefully implanted rostrally, aiming its tip just dorsal to the lumbar enlargement. The position of the catheter was checked postmortem.
2.5. Behavioral testing, paw withdrawal frequency
The animals were placed on a metal mesh in a transparent plastic box and were habituated to the testing environment for at least 30min before testing began. The effects of PI3K inhibitors and PKB/Akt inhibitors on capsaicin-induced mechanical hypersensitivity were observed by testing the paw withdrawal frequency following sequential applications of three von Frey filaments with bending forces of 30, 100 and 200mN to the base of the middle toe before and after intradermal injection of capsaicin (Tu et al., 2004). The distance between the capsaicin injection site and the testing site was about 2cm. After adaptation to the testing environment, animals were tested three times at 10min intervals as a control before administration of any drug. A trial consisted of five mechanical stimuli at intervals of 2s, and such trials were repeated at 10min intervals in the first hour, at 20min intervals in the second hour, and at 30min intervals in the third hour after capsaicin (0.1%, 10μl) injection. In each trial, the first two responses were discarded to exclude the effects of animal alarm; otherwise, each withdrawal of the paw during the application of the mechanical stimuli was considered a response. All behavioral experiments were started in the morning between 8 am and 9 am and finished by noon or in the early afternoon. Statistical significance was determined by using a Kruskal-Wallis test.
2.6. Rotarod performance in rats
To determine the effects of the compounds on motor co-ordination, animals were first trained on a treadmill (Ugo Basile Rota-Rod, Stoelting, Wood Dale, IL) for 2 days before the testing, which included three sessions of three intervals given daily with an intersession interval >2h and an intertrial interval >5min. Animals then received drug treatments and 20min later were placed on an accelerating rotarod (increasing from 5 to 40rpm during a 10min period) and the time during which the rats were able to remain on the rotarod was recorded up to a cut off of 10min. Values are presented as mean±SEM. Comparison between the means was analyzed with ANOVA followed by a Newman-Keuls test.
3. Results
3.1. Activation of PKB/Akt in spinal dorsal horn by intradermal injection of capsaicin
The distribution of p-PKB/Akt in the spinal cord was observed. We used an antibody specific for phosphorylated serine 473, which is essential for maximal activation of PKB/Akt (Alessi et al., 1996). In control animals, although p-PKB/Akt-immunoreative staining could be detected in all of the spinal cord laminae, it was distributed predominantly in laminae I–IV, especially in laminae I–II (Fig. 1A and C). Five minutes after unilateral intradermal injection of capsaicin in the hindpaw, staining of p-PKB/Akt increased robustly in the ipsilateral dorsal horn. The most prominent increase was found in laminae I–II (Fig. 1B and D). In order to identify the cell types that expressed p-PKB/Akt, double immunostaining of p-PKB/Akt (Fig. 2A) and the neuronal marker, neuronal-specific nuclear protein (NeuN) (Fig. 2B), was performed. p-PKB/Akt colocalizes with NeuN in the spinal cord after capsaicin stimulation (Fig. 2C). However, we also found a large amount of background p-PKB/Akt staining (Figs. 1A–D, 2A and C). This suggests that p-PKB/Akt might also be localized in central terminals of primary sensory afferents, other synaptic endings or glia cells. These results indicate that PKB/Akt is activated in spinal cord after capsaicin stimulation.
Increase in p-PKB/Akt in dorsal horn following intradermal injection of capsaicin. (A) immunofluorescence imaging showing the expression of p-PKB/Akt in the contralateral and ipsilateral dorsal horn of a vehicle injected control rat. p-PKB/Akt-immunoreative staining could be detected in all of the spinal cord laminae (Fig. 1A). (B) the increased expression of p-PKB/Akt in the contralateral and ipsilateral dorsal horn induced by intradermal capsaicin injection. Five minutes after unilateral intradermal injection of capsaicin in the hindpaw, staining of p-PKB/Akt increased robustly in the ipsilateral dorsal horn (Fig. 1B). It is distributed predominantely in laminae I–IV, especially in laminae I–II (Fig. 1A and C). The most prominent increase was found in laminae I–II (Fig. 1B and D). Higher magnification of the immunofluorescence confocal images shown in (C and D) demonstrates the expression of p-PKB/Akt-immunoreative cells in laminae of dorsal horn. In control rats, it is distributed predominantely in laminae I–IV, especially in laminae I–II (Fig. 1C). In capsaicin injected rats, the number of p-PKB/Akt-immunoreative cells increased significantly in the ipsilateral dorsal horn. The most prominent increase was found in laminae I–II (Fig. 1D).
Increase in p-PKB/Akt in dorsal horn neurons 5 min after intradermal injection of capsaicin. Representative confocal images were obtained from slices that were double labeled using antibodies specific for p-PKB/Akt and neuronal-specific nuclear protein (NeuN). p-PKB/Akt labeling is indicated by red (A), NeuN labeling is indicated by green (B), and dual labeling is indicated by yellow (C).
To study the time course of changes in PKB/Akt activation, Western blots were performed. p-PKB/Akt was detected in the dorsal part of the spinal cord (at L4–6). Four groups of rats were used: control (vehicle), and 5, 30 and 60min after unilateral intradermal injection of capsaicin. p-PKB/Akt expression increased significantly in the dorsal spinal cord by 5min after the intradermal capsaicin injection (Fig. 3A and B). p-PKB/Akt was still significantly higher at 30min, and this increase lasted at least 60min after capsaicin injection (Fig. 3B).
Western blots reveal an increased expression of p-PKB/Akt in the dorsal spinal cord at various times following intradermal injection of capsaicin. (A) Representative Western blots for p-PKB/Akt and β-actin. β-actin was used as loading control. Con, control; 5, 30, 60 are minutes after capsaicin injection. (B) p-PKB/Akt immunoreactivity was normalized to β-actin immunoreactivity. Values are expressed as percentage of control, mean±SEM. p-PKB/Akt was increased by 5 min following capsaicin injection, was still increased at 30 min, and this increase lasted at least 60 min after capsaicin injection. *P<0.05, statistical significance compared with control (0 time point) with one-way ANOVA followed by Newman-Keuls test (_n_=7).
4. PKB/Akt signaling pathway mediates capsaicin-induced mechanical hypersensitivity
Intradermal injection of capsaicin induced mechanical hypersensitivity, a sign of central sensitization. The mechanical hypersensitivity was indicated by an enhanced paw withdrawal frequency to application of von Frey filaments with different bending forces. Three von Frey filaments with bending forces of 30, 100 and 200mN were used to test the paw withdrawal frequency. The paw withdrawal frequency was significantly increased in a mechanical stimulation intensity-dependent manner after capsaicin injection (Fig. 4A). The paw withdrawal frequency to application of von Frey filaments with 30, 100 and 200mN bending forces was increased significantly by 10min and the increase lasted at least 30min for 30mN, 80min for 100mN, and 100min for 200mN after capsaicin injection (Fig. 4A). To test whether the PKB/Akt signaling pathway is involved in capsaicin-induced mechanical hypersensitivity, the PI3K (upstream of PKB/Akt) inhibitors, wortmannin and LY294002, and the PKB/Akt inhibitors, NL-71-101, SH-6, IV and V, were injected intrathecally in different groups of rats. Post-treatment and pre-treatment paradigms were applied. In pre-treatment experiments, all inhibitors were applied intrathecally 20min before capsaicin injection, and the mean paw withdrawal frequency, i.e. mean area under the curve (MAUC), within 10–60min after capsaicin to 30, 100 and 200mN von Frey filaments was used to evaluate the effect of the inhibitors in preventing the development of hypersensitivity. Wortmannin (0.4μg/10μl) and LY294002 (2.5μg/10μl) pre-treatment inhibited the enhanced paw withdrawal frequency to 30, 100 and 200mN von Frey filaments induced by capsaicin (Fig. 4A and B). The PKB/Akt inhibitor SH-6 (0.006μg/10μl, 0.06μg/10μl, 0.6μg/10μl) dose dependently inhibited the secondary mechanical hypersensitivity(Fig. 5a1–a3). PKB/Akt inhibitor IV (0.06μg/10μl, 0.6μg/10μl) also blocked the mechanical hypersensitivity in a dose dependent manner (Fig. 5b1–b3). Furthermore, another recently designed potent agent, PKB/Akt inhibitor V (0.3μg/10μl), also prevented the mechanical hypersensitivity (Fig. 5b1–b3). In summary, the pre-treatment studies indicated that the PKB/Akt pathway mediates the initiation of mechanical hypersensitivity induced by capsaicin.
Pre-treatment with PI3K inhibitors and PKB/Akt inhibitors prevents the mechanical hypersensitivity induced by capsaicin. (A) Time course of mechanical hypersensivity induced by capsaicin, and time course after pre-treatment with PI3Kinhibitors wortmannin (0.4 μg/10 μl) and LY294002 (2.5 μg/10 μl). The mechanical hypersensitivity is shown by the enhanced paw withdrawal frequency to applications of von Frey filaments with different bending forces (30, 100, 200 mN). Wortmannin or LY294002 was injected 20 min before capsaicin (0.1%, 10 μl). *P<0.05, #P<0.05, $P<0.05, significant difference from time point (0 time point) before capsaicin, respectively, in 200 mN vehicle, 100 mN vehicle, 30 mN vehicle line. (B) Pre-treatment with wortmannin and LY294002 inhibit the mechanical hypersensivity. The mean paw withdrawal frequency, i.e. the mean area under the curve (MAUC) within 10–60 min after capsaicin to 30, 100 and 200 mN von Frey filaments was used to evaluate the effect. **P<0.01, ***P<0.001, significant difference compared with vehicle (10% DMSO) group (_n_=5–6).
Pre-treatment with PKB/Akt inhibitors blocks the mechanical hypersensitivity. (a1–a3) SH-6 (0.006, 0.06, 0.6 μg/10 μl) dose dependently inhibits mechanical hypersensitivity.*P<0.05, **P<0.01, ***P<0.001, significant difference compared with vehicle (10% DMSO) group (_n_=6). (b1–b3) PKB/Akt inhibitor IV (0.06, 0.6 μg/10 μl) blocks the hypersensitivity in dose-dependent manner. Inhibitor V (0.3 μg/10 μl) also had the same effect. **P<0.01, ***P<0.001, significant difference compared with vehicle(10% DMSO) group (_n_=5–7).
To investigate whether the PKB/Akt pathway is involved in the maintenance of mechanical hypersensitivity, PI3K and PKB/Akt inhibitors were injected intrathecally 30min after capsaicin injection. The mean paw withdrawal frequency within 40–100min after capsaicin to 100 and 200mN von Frey filaments was used to evaluate the effect of the inhibitors in blocking the established hypersensitivity. Wortmannin (0.04μg/10μl, 0.4μg/10μl) reversed the established mechanical hypersensitivity in a dose dependent manner (Fig. 6C and D). However, LY (2.5μg/10μl) had no effect on the established mechanical hypersensitivity, although the same dose of LY294002 (2.5μg/10μl) applied before capsaicin prevented the mechanical hypersensitivity (Fig. 4A and B). Concerned that a higher concentration of DMSO might have some side effect on the behavior, we did not test a higher dose of LY294002. The PKB/Akt inhibitor NL-71-101 (also inhibits PKA) (0.01/10μl, 0.1/10μl, 1μg/10μl) inhibited the mechanical hypersensitivity in a dose-dependent manner (data not shown). The PKB/Akt inhibitors SH-6 (0.6μg/10μl), IV (0.6μg/10μl) and V (0.3μg/10μl), also blocked the established mechanical hypersensitivity induced by capsaicin (Fig. 6A–D). The results indicate that the PKB/Akt pathway mediates both the initiation and expression of mechanical hypersensitivity. Moreover, the results imply that the PKB/Akt pathway contributes to pain hypersensitivity induced by intradermal injection of capsaicin.
Post-treatment with PI3K inhibitors or PKB/Akt inhibitors blocked the established mechanical hypersensitivity induced by capsaicin. (A and B), Time course of post-treatment with PKB/Akt inhibitors SH-6 (0.6 μg/10 μl), inhibitor IV (0.6 μg/10 μl) or V (0.3 μg/10 μl) inhibiting mechanical hypersensitivity. (C and D) Post-treatment with PI3K or PKB/Akt inhibitors inhibit the established mechanical hypersensitivity. The mean paw withdrawal frequency, i.e. MAUC within 40–100 min after capsaicin to 100 and 200 mN von Frey filaments was used to evaluate the effect. Wortmannin (0.04, 0.4 μg/10 μl), LY294002 (2.5 μg/10 μl), SH-6 (0.6 μg/10 μl), inhibitor IV (0.6 μg/10 μl) or V(0.3 μg/10 μl) were applied 30 min after capsaicin injection. *P<0.05, **P<0.01, ***P<0.001, significant difference compared with vehicle (10% DMSO) group (_n_=5–7).
Inhibition of p-PKB/Akt activation in the spinal cord by PI3K inhibitors or PKB/Akt inhibitors at a dose that affects behavior was confirmed by Western blots (Fig. 7A and B). Wortmannin (0.4μg/10μl) or SH-6(0.6μg/10μl) was applied intrathecally 20min before capsaicin (0.1%, 15μl) injection. We found that intrathecal injection of wortmannin or SH-6 at a dose that affects behavior significantly suppressed the increase of p-PKB/Akt in the dorsal spinal cord at 5min after capsaicin injection, compared to vehicle (10% DMSO)-treated rats (Fig. 7A and B). However, there is no significant difference in the immunoreactivity for p-Akt between control (vehicle) and wortmannin groups (Fig. 7A and B). These results further confirm that activation of the PKB/Akt signaling pathway mediates capsaicin-induced mechanical hypersensitivity.
Western blots show PI3K and PKB/Akt inhibitors block the increased expression of p-PKB/Akt in the dorsal spinal cord following intradermal injection of capsaicin. (A) Representative Western blots for p-PKB/Akt and β-actin. β-actin was used as loading control. V+CAP, vehicle+capsaicin; W+CAP, wortmannin+capsaicin; SH-6+CAP, SH-6+capsaicin; W, wortmannin. Vehicle, wortmannin or SH-6 at behavioral dose was injected intrathecally 20 min before capsaicin injection. (B) p-PKB/Akt immunoreactivity was normalized to β-actin immunoreactivity. Values are expressed as percentage of control, mean±SEM. There is no significant difference in the immunoreactivity for p-Akt between control (vehicle) and wortmannin groups. p-PKB/Akt was increased following capsaicin injection in V+CAP group compared to control group. However, p-PKB/Akt was not changed significantly following capsaicin injection in W+CAP or SH-6+CAP group. *P<0.05, statistical significance compared with control, #P<0.05, statistical significance compared with V+CAP group with one-way ANOVA followed by Newman-Keuls test (_n_=4–7).
To examine if the animal's motor ability was affected by intrathecal administration of PI3K or PKB/Akt inhibitors, rotarod performance was observed. Intrathecal (i.t.) catheters were implanted in rats before behavioral testing. Seven days later, the rats were trained on the computerized treadmill for 2 days, then wortmannin (0.4μg/10μl), SH-6 (0.6μg/10μl) or vehicle (10% DMSO) were injected intrathecally in three groups of rats separately. Twenty minutes later, the mean running time of the rats was tested. There were no significant changes in mean running time among naïve rats, rats with an intrathecal catheter, or the vehicle, wormannin and SH-6 groups (Fig. 8). The results indicate that under our experimental conditions, motor function was unaffected by intrathecal administration of either wortmannin or SH-6.
Lack of effect of PI3K or PKB/Akt inhibitor on motor ability as shown by the rotarod test. Mean running time in seconds is shown on the ordinate. The bars are for naïve rats; the control group (rats with intrathecal catheter); the vehicle group (rats given 10% DMSO intrathecally); the wortmannin group (rats that received wortmannin (0.4 μg/10 μl)); and the SH-6 group (rats that received SH-6 (0.6 μg/10 μl)). Intrathecal injections were given 20 min before the rotarod test was started. One way ANOVA was performed to compare the difference between groups.
5. Discussion
Continuous activation of peripheral afferent fibers by noxious stimulation induces the release of excitatory neurotransmitters into the dorsal horn, and these may elicit pain hypersensitivity (central sensitization). The central sensitization is triggered by the activation of post-synaptic excitatory receptors in dorsal horn and mediated by several signaling pathways, including PKA, PKC, PKG, CAMKII and ERK.
The Akt family of serine/threonine protein kinases is activated by growth factors (Kaplan and Miller, 2000), as well as by a variety of physiological stimuli (Brazil et al., 2002; Datta et al., 1999; Downward, 1998; Kandel and Hay, 1999; Meier and Hemmings, 1999; Scheid and Woodgett, 2003; Vanhaesebroeck and Alessi, 2000). Akt kinases are members of the superfamily of protein kinases that includes PKA and PKC, leading to the alternative name for AKT of PKB (Kandel and Hay, 1999). PKB/Akt has been implicated in a variety of cellular processes, including glucose metabolism, transcription, apoptosis, proliferation, migration and angiogenesis (Brazil and Hemmings, 2001). Growing evidence indicates that PI3K is a lipid kinase that phosphorylates the D-3 position of phosphatidylinositol lipids to produce PI (3,4,5), which acts as a membrane-embedded secondary messenger to recruit and activate Akt/PKB (Chan et al., 1999; Franke et al., 1997). PKB/Akt activation is regulated primarily through phosphorylation of Thr-308 or Ser-473 by phosphoinositide-dependent kinases. In the nervous system, the PI3K-PKB/Akt signaling pathway is activated by trophic factors, growth factors, hormones, or neurotransmitters, and participates in cellular activity that underlies development (Ivanova et al., 2002; Markus et al., 2002; Meier and Hemmings, 1999; Sutton and Chandler, 2002; Znamensky et al., 2003). Ample and growing evidence indicates that the PI3K-PKB/Akt pathway is involved in synaptic plasticity such as long-term potentiation (LTP), long-term depression (LTD) (Hou and Klann, 2004; Kelly and Lynch, 2000; Opazo et al., 2003; Sanna et al., 2002) and brain-derived neurotrophic factor (BDNF)-dependent spatial memory formation (Mizuno et al., 2003). Recently, it has been reported that PI3K mediates the primary heat hyperalgesia induced by capsaicin or by NGF (Zhuang et al., 2004) and there is an activity-dependent phosphorylation of PKB/Akt in adult DRG neurons (Pezet et al., 2004, 2005). However, whether PKB/Akt contributes to spinal cord central sensitization is poorly understood. In this study, we found that p-PKB/Akt is distributed in the spinal cord in normal rats. Furthermore, p-PKB/Akt is significantly increased after intadermal injection of capsaicin. The increase peaks by 5min after capsaicin injection and lasts at least 60min. Moreover, the enhanced p-PKB/Akt is predominantly distributed in neurons of laminae I–II. This indicates that PKB/Akt is activated in neurons of the superficial spinal dorsal horn by strong noxious stimulation.
Our behavioral experiments demonstrated that the PKB/Akt pathway mediates capsaicin-induced mechanical hypersensitivity. We used two different kinds of PI3K (upstream of PKB/Akt) inhibitors, wortmannin and LY294002, and four different kinds of PKB/Akt inhibitors, NL-71-101, SH-6, PKB/Akt inhibitor IV and V. Wortmannin and LY294002 are structurally unrelated PI3K inhibitors. The four PKB/Akt inhibitors also have different structures and different target sites for their actions (Kau et al., 2003; Kozikowski et al., 2003; Reuveni et al., 2002; Yang et al., 2004). Pre-treatment with wortmannin, LY294002, SH-6, inhibitor IV or V prevents the development of mechanical hypersensitivity induced by capsaicin. Post-treatment with wortmannin, NL-71-101, SH-6, inhibitor IV or V also blocks the established mechanical hypersensitivity. However, a low dose of LY294002 that can prevent hypersensitivity by pre-treatment does not block the hypersensitivity by post-treatment. Furthermore, pre-treatment with other PI3K or PKB/Akt inhibitors is also more effective than post-treatment. The different effects of wortmannin and LY294002 in post-treatment might be due to the doses of wortmannin and LY294002 we used. The IC50 for LY294002 (1.4μM) is 280 times of that for wortmannin (5nM). In this study, the concentration of LY294002 (8×10−4M, 2.5μg/10μl) is only eight times of that of wortmannin (10−4M, 0.4μg/10μl). This might be the reason for their different effects in post-treatment experiments. Wortmannin and LY294002 are structurally unrelated, as are the PKB/Akt inhibitors used, and the PKB/Akt inhibitors work on different targets; yet all of these drugs inhibit the mechanical hypersensitivity induced by capsaicin. Therefore, we conclude that the PKB/Akt pathway helps mediate the mechanical hypersensitivity induced by capsaicin.
It should be mentioned that the rats did not respond to the three von Frey filaments with bending forces of 30, 100 and 200mN before injection of capsaicin (Fig. 4A). Therefore, we could not detect whether the Akt inhibitors affected basal mechanical pain responses at these strengths. However, the rats did respond to the two higher forces of punctate stimuli (320 and 450mN) (data not shown). We think the stronger punctate stimuli are painful mechanical stimuli. The inhibitor (Akt inhibitor IV) did inhibit the nociceptive responses induced by these stronger stimuli. After injection of capsaicin, the rats became responsive to the three weaker von Frey filaments (30, 100, and 200mN). This suggests that intradermal injection of capsaicin induced mechanical hypersensitivity. The PI3K-PKB/Akt inhibitors inhibited this mechanical hypersensitivity (Figs. 4–6). It may be questioned if Akt inhibition had inhibitory effect on nociception and that it had no effect per se on mechanical hypersensitivity. However, we found that Akt inhibition had no effect on the baseline activity of WDR neurons induced by brush (brushing the skin with a camel hair brush) or press (which produced a sensation of firm pressure, 144g/mm2 near the threshold for pain). Moreover, it significantly inhibited the hyperactivity of WDR neurons to brush and press induced by intradermal injection of capsaicin (Sun et al., 2004a,b,c and our unpublished data). We also found that there was no effect of wortmannin at the dose used in this study on basal Akt phosphorylation (Fig. 7A and B; the densities of the p-Akt bands normalized with respect to that of β-Actin, are 1.14±0.07, 1.10±0.21 in vehicle and wortmannin treated groups, respectively, _n_=4–7). Based on the electrophysiological and Western blot experiments, we conclude that the effect of Akt inhibitors at the doses used in this study on the mechanical hyperactivity induced by noxious stimuli is not due to its effect on basal nociception.
In this study, we cannot conclude that hypersensitivity is only due to a central spinal cord mechanism, because Serra et al. (2004) found that a majority of mechano-insensitive (CMi) nociceptors in human skin developed responsiveness to mechanical stimuli after capsaicin injection, and this sensitization may contribute to hypersensitivity. Another question is that even if the hypersensitivity is only due to a central spinal cord mechanism, the PKB inhibitor still can affect activation of PKB/Akt in DRG through a dorsal root reflex mechanism. Furthermore, intrathecal injection of inhibitors perhaps not only acts in the spinal cord, but also in the DRG. However, Western blots showed that injection of 0.1% capsaicin (15μl, dose used for behavioral experiments in this study) did not induce an increase of p-PKB/Akt in DRG (L4–6) (data not shown), although 1% capsaicin (50μl) injection did (our unpublished data). Most importantly, p-PKB/Akt in lumber spinal cord increased significantly after a capsaicin (0.1%, 15μl) injection, and PI3K or PKB/Akt inhibitors could block the increase. In our electrophysiological experiments, we used the microdialysis technique to deliver the PKB/Akt inhibitor SH-6. The SH-6 was delivered locally to the spinal cord by using this technique. We found that SH-6 significantly inhibited the hyperactivity of wide dynamic range (WDR) induced by intradermal injection of capsaicin (Sun et al., 2004a,b,c). The results suggest that the hypersensitivity is at least partly dependent on a central mechanism.
Pre-synaptic and post-synaptic NMDA receptors have very important roles in peripheral and central sensitization (Du et al., 2003; Guo et al., 2002; Zou et al., 2000). Pre-synaptic NMDA receptors and their stimulation might be responsible for the mechanical hypersensitivity via a retrograde sensitization of the primary sensory neurons. In cultured cortical neurons, PKB activity was associated with NMDA receptor activation. Thus, PKB activity may be associated with NMDA receptor (pre-synaptic and post-synaptic) activation in spinal cord. The effect of PKB on NMDA receptors could be pre-synaptic, post-synaptic or both. Therefore, the effect of the PKB inhibitors could reflect an indirect effect of PKB by acting on pre-synaptic NMDA receptors in this study.
The molecular pathways responsible for PKB/Akt activation and the downstream actions of PKB/Akt phosphorylation are not clear as yet. We hypothesize that activation of corresponding NMDA receptors, metabotropic glutamate receptors and tyrosine receptor kinase B (TrkB) receptors activates the PI3K-PKB/Akt pathway in dorsal horn neurons. The downstream action of PKB/Akt is involved in transcription and protein synthesis in brain and cultured neurons (Hou and Klann, 2004; Perkinton et al., 2002; Rodgers and Theibert, 2002). In this study, the key effect of PKB/Akt activation in pain hypersensitivity must be by non-transcriptional means, because the latency for the action of PKB/Akt inhibitors was very short. Thus, the downstream effects of PKB/Akt in pain hypersensitivity include translational and post-translational modifications of kinases, receptors and ion channels.
In conclusion, activation of the PKB/Akt signaling pathway contributes to the mechanical hypersensitivity induced by capsaicin. This implies that inhibitors of the PKB/Akt pathway have potential for therapies to reduce pain hypersensitivity.
Acknowledgements
We thank XiaoJu Zou, Lingfei Hou and Zaiming Ye for technical support. This work was supported by NIH grants NS09743 and NS11255.
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Keywords:
Phosphorylation; Dorsal horn; Protein kinase; Capsaicin; Pain; Phosphoinositide 3-kinase
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