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NEUROPHARMACOLOGY

Protein Kinase C and : Involvement in Formalin-Induced Nociception in Neonatal Rats

Departments of Anesthesia (S.M.S., S.M.E.W., M.C.P., D.C.Y., J.J.K.) and Molecular Pharmacology (D.M.-R.), Stanford University School of Medicine, Stanford, California

Received for publication September 19, 2003
Accepted February 3, 2004.

	Abstract

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The central nervous system undergoes dynamic changes as it matures. However, until recently, very little was known about the impact of these changes on pain and analgesia. This study tested the hypothesis that the and isozymes of protein kinase C (PKC) contribute to formalin-induced nociception in an age-dependent manner. Expression of and PKC and the contributions of these isozymes in formalin-induced nociception was examined in postnatal day 7, 15, and 21 rats. PKC expression in dorsal root ganglion neurons and PKC expression in lamina II of the spinal cord increased from the first to the third postnatal week. Coupling immunohistochemical and Western analysis, translocation of PKC followed intraplantar formalin in all ages. In contrast, formalin-induced PKC translocation was observed only in postnatal day 21 rats. Behaviorally, intrathecal administration of the PKC-specific inhibitor (V1-2) attenuated phase 1 and phase 2 formalin behaviors at all ages. In contrast, intrathecal administration of the PKC-specific inhibitor (V5-3) attenuated only phase 2 responses in postnatal day 15 and 21 rats. Functionally, inhibition of PKC decreased capsaicin-stimulated release of glutamate and calcitonin gene-related peptide in spinal cords isolated from postnatal day 7 rats. These results suggest that PKC age independently mediates inflammatory pain produced by intraplantar formalin. In contrast, PKC contributes to formalin-induced nociception in an age-dependent manner. Identifying the molecular mechanisms responsible for age-specific patterns of nociception is necessary for the rational development of novel therapeutic strategies for treating pediatric pain.

Nonspecific inhibitors of protein kinase C (PKC) attenuate phase 2 formalin-induced pain behaviors in adult animals (Yashpal et al., 1995). Increased activation and translocation of PKC in dorsal horn neurons has been reported after peripheral administration of formalin (Yashpal et al., 1995). PKC is an important family of signal transduction molecules that function to phosphorylate a wide range of intracellular proteins. The PKC gene family has been divided into three groups of isozymes based on sequence homology and biochemistry: calcium- and diacylglycerol (DAG)-dependent PKC isozymes (, I, II, and ), Ca²⁺-independent but DAG-dependent PKC isozymes (, , , and ), and Ca²⁺- and DAG-independent PKC isozymes. In adult rats, PKC has been identified in the superficial laminae of the dorsal spinal cord (Polgar et al., 1999) whereas PKC has been identified in adult rat primary afferents that terminate in the superficial dorsal horn (Khasar et al., 1999). In addition to the presence of and PKC in anatomical sites important for pain processing, a functional role for these two isozymes in pain processing in adult rodents has been identified using knockout mouse technology (Malmberg et al., 1997; Khasar et al., 1999).

Expression and activity of some PKC isozymes are developmentally regulated (Hashimoto et al., 1988; Akinori, 1998). The impact of age-dependent changes in PKC isozymes on pain processing is not known. PKC translocation requires the binding of PKC to isozyme-selective docking proteins, receptors for activated C kinase (RACK) (Mochly-Rosen and Gordon, 1998). Isozyme-selective PKC inhibitor peptides compete with activated PKCs for binding to RACK, thus preventing translocation and activity of the corresponding PKC isozymes (Mochly-Rosen and Gordon, 1998). Linking of isozyme-specific PKC inhibitor peptides to cell-permeable carrier peptides (e.g., derived from Tat, antenapedia, or even a poly arginine) enables efficient transfer of inhibitor peptides into cells and tissue (Chen et al., 2001). Upon entering the cell, the disulfide bond linking carrier to peptide is reduced, releasing the inhibitor peptide inside the cell to interact with its corresponding RACK (Chen et al., 2001). In the present study, isozyme-specific peptide inhibitors were used to determine whether intraplantar formalin induces translocation of and PKC in an age-specific manner and whether this translocation mediates nociception.

	Materials and Methods

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Animals. P7, P15, and P21 male and female Sprague-Dawley rats (Simenson Laboratories, Gilroy, CA) were housed with their dam in a 12/12-h light/dark cycle (lights on at 7:00 AM) with food and water available ad libitum. All experimental protocols were approved by the Institutional Animal Care and Use Committee at Stanford University. Efforts were made throughout the experiment to minimize animal discomfort and to reduce the number of animals used.

Peptide Synthesis. All peptides were synthesized at Stanford's Protein and Nuclei Acid facility and conjugated to Tat, amino acids 47–57 [YGRKKRRQRRR] via a cysteine-cysteine bond at their N termini. The PKC antagonist V1-2 [EAVSLKPT] and the PKC antagonist V5-3 [CRLVLASC] were used at >90% purity.

Experimental Design. Spinal drug delivery in neonatal rats poses a unique challenge based on the size of the neonatal rats and the rapid elongation of the vertebral column and spinal cord during the first few postnatal weeks. These factors make insertion of a spinal catheter prohibitive or at the very least extremely difficult. Thus, we administered PKC peptide inhibitors reversibly linked to Tat carrier protein (5 µl in P7 rats, 10 µl in P15 and P21 rats) via direct lumbar puncture. The volume necessary to deliver the peptides to the lumbar spinal cord was determined previously by using Evan's blue dye to determine that the spread of fluid reached the lumbar spinal cord. The increasing length and volume of the spinal cord between P7 and P21 required a larger volume of the same concentration to be delivered in the older rat. Thus, although twice as much compound is being delivered in P21 rats compared with P7 rats the increase in the surface area being covered by that volume ensures that the same anatomical region of spinal cord is seeing equivalent concentrations of drug (Marsh et al., 1999). Intrathecal drug delivery was completed by inserting a sterile 29-gauge 3/10-ml insulin syringe between the S1 and L6 vertebrae in rats anesthetized with halothane.

Tat-conjugated peptide inhibitors specific to the (V1-2) and (V3-5) isoforms of PKC, Tat carrier alone, or saline vehicle were administered 15 min before intraplantar formalin (1% in 10 µl in P7 pups, 2.5% in 10 µl in P15 and P21 pups; n = 6–10/group) or saline. Peptides were administered at 10, 50, or 100 pmol/5 µl. Rats were placed in Plexiglas observation boxes (3.5 inches in width x 6 inches in length x 8 inches in height), and behaviors were observed for 1 h. In all experiments, animals were maintained at nesting temperature with a heating pad placed below the observation chambers. The time sampling method for behavioral observation was used, in which the observer rapidly records the behavior of the animals every 2 min (Teng and Abbott, 1998). A score of 1 is given if the animal is licking, shaking, or elevating its hindpaw. A 6-min period of observation provides a maximum pain score of 3 for each animal. All studies were completed with the experimenter blinded to treatment group.

Immunohistochemistry. P7, P15, and P21 rats were administered 50 pmol/5 µl of Tat carrier, V1-2, or V3-5 intrathecal 15 min before intraplantar formalin (1% in 10 µl in P7 rats, 2.5% in 10 µl in P15 and P21 rats) or saline as described above in the behavioral experiments. At 5 and 10 min postformalin or saline, rats were deeply anesthetized and underwent transcardiac perfusion (n = 3/treatment/time). Lumbar spinal cord and L5 dorsal root ganglia (DRG) were harvested and fixed in 4% paraformaldehyde for 3 h. Tissue was cryoprotected in 30% sucrose at 4°C for a minimum of 48 h. Sections were freeze-mounted in OCT embedding medium on cork blocks for cryostat sectioning. For spinal cord sections, free-floating immunohistochemistry was performed on 30-µm L4–L5 spinal sections. DRG were cut on a cryostat at 10 µm and slide mounted. Slides were heated overnight at 32°C. Immunohistochemistry was performed using an avidin-biotin complex technique as described previously (Sweitzer et al., 1999). Rabbit polyclonal antibodies to PKC (1:500; cPKC and sc-211) or PKC (1:1000; nPKC and sc-214) from Santa Cruz Biotechnology (Santa Cruz, CA) were used.

Immunohistochemistry was scored blinded to experimental conditions. PKC immunoreactive DRGs and PKC immunoreactive lumbar spinal cords were imaged with a digital camera, and the images were transferred to a computer for analysis of cell counts and/or densitometry. All data represents the average from at least three tissue sections/animal with a minimum of three animals per group. Mean density of PKC immunoreactivity per DRG section was calculated using ImageJ software. Density was normalized to tissue staining levels in age-matched normal naive rats using the following calculation: (TRT_TD - TRT_BD)/(NORM_TD - NORM_BD) x 100. TRT is the staining density in the tissue from the treated rat, and NORM is the staining density in the tissue from normal naive rats. TD represents the total staining density of the tissue, and BD is the background staining density.

Western Analysis. Identical to the immunohistochemical analysis, P7 and P21 rats were administered 50 pmol/5 µl of Tat carrier, V1-2, or V3-5 intrathecal 15 min before intraplantar formalin (1% in 10 µl in P7 rats, 2.5% in 10 µl in P21 rats) or saline. At 5 and 10 min postformalin or saline, left lumbar spinal cord and left L4–L6 DRG were harvested and frozen on dry ice after CO₂ asphyxiation and decaptitation (n = 3/time/treatment). DRG and spinal cord tissues were homogenized and sonicated in lysis buffer (10 mM HEPES, 42 mM KCl, 5 mM MgCl₂, 0.1 mM EDTA, 0.1 mM EGTA, and 1% Triton X-100) supplemented with protease inhibitors (Complete Mini; Roche Diagnostics, Mannheim, Germany). Proteins were quantitated using the BCA protein assay (Pierce Chemical, Rockford, IL), and 12 or 18 µg of protein (DRG or spinal cord, respectively) in SDS loading buffer was resolved on 7.5% SDS polyacrylamide gels. After protein transfer, polyvinylidene difluoride membranes were blocked in Tween 20/PBS containing 5% powdered milk for 1 h at room temperature. Membranes were then blotted with nPKC (1:200; sc-214), or cPKC (1:200; sc-211) (Santa Cruz Biotechnology) overnight at 4°C in Tween 20/PBS containing 5% powdered milk. Membranes were then incubated with horseradish peroxidase-conjugated goat anti-rabbit antibody (1:2500) (Jackson ImmunoResearch Laboratories Inc., West Grove, PA), and bands were detected using enhanced chemiluminescence detection (Amersham Biosciences UK, Ltd., Buckinghamshire, Little Chalfont, UK). Membranes were then blotted with -actin (1:5000; Sigma-Aldrich) for2hat room temperature in Tween 20/PBS containing 5% powdered milk. Membranes were then incubated with horseradish peroxidase-conjugated anti-mouse antibody (1:2500) (Cell Signaling Technology Inc., Beverly, MA) and bands were detected using enhanced chemiluminescence detection (Amersham Pharmacia Biotech, Buckinghamshire, England).

Calcitonin Gene-Related Peptide (CGRP) and Glutamate Release Assays. Whole spinal cords from P7 rats were isolated and maintained in oxygenated artificial cerebrospinal fluid for 1 h before experimental manipulation. Release assays using whole isolated spinal cords were only studied in P7 rats. Our previous experience suggests limited viability of this whole cord preparation prepared from older rats. The dose of capsaicin required to elicit maximal release of CGRP was determined by incubating the spinal cords in 800 µl of artificial cerebrospinal fluid to serve as baseline control release. A second 10-min incubation was done in the presence of 0, 2.5, 25, and 100 µM capsaicin (n = 4/treatment). To determine the dependence on extracellular calcium isolated cords were maintained in artificial cerebrospinal fluid lacking Ca²⁺ throughout the equilibration period as well as during the baseline control release and capsaicin (25 µM)-stimulated release.

The broad-spectrum PKC inhibitor GF109203X (1.2 µM; Sigma-Aldrich), the calcium-dependent PKC inhibitor Go6976 (1 µM; Calbiochem, San Diego, CA), the PKC-specific peptide inhibitor (V1-2; 25.6 pmol), Tat carrier (25.6 pmol), or saline vehicle was introduced in 800 µl of artificial cerebrospinal fluid for 10 min (n = 4/treatment). This fraction was collected and served as baseline control release. The concentration of PKC inhibitors is based on other studies in this laboratory showing efficacy at this concentration in preventing naloxone precipitated hyperresponsiveness of the slow ventral root potential after morphine exposure in P7 spinal cord (Sweitzer et al., 2004). Capsaicin (25 µM) was introduced in the presence of either V1-2, Tat carrier, or saline vehicle and incubated with the isolated cords for 10 min.

Analysis of CGRP by Enzyme-Linked Immunosorbent Assay. Samples were analyzed using the rat CGRP enzyme immunoassay kit (SPI-Bio, Massy Cedex, France). Each sample was performed in duplicate as directed in the instructions. Briefly, each plate was washed three times with 300 µl of wash buffer before adding 100 µl of either sample, standard, or buffer to each well. In addition, each well received 100 µl of anti-CGRP acetylcholinesterase tracer. Samples were incubated overnight at 4°C with agitation. The next day, samples were washed three times with 300 µl of wash buffer, before adding 200 µl of Ellman's reagent. Absorbances were recorded at 405 nm at 30 min. CGRP concentrations were determined by reference of the sample absorbances to the standard curve generated off the recombinant rat CGRP supplied with the kit.

Analysis of Glutamate by High-Performance Liquid Chromatography. Methods were derived from Graser et al. (1985). Samples (20 µl) were reacted with 1.0 µl of o-phthaldialdehyde/3-mercaptoproprionic acid reagent (Sigma-Aldrich) for derivitization. After 1 min, a 20-µl sample was injected into the high-performance liquid chromatograph, which uses a reversed-phase C18 column (3.9 x 150 mm; Waters, Bedford, MA). Glutamic acid was quantitated at absorbance of 340 nm (Waters 2487). The samples were separated with a mobile phase gradient using the following solutions: 12.5 mM NaPO₄, pH 7.2, and a 50% (v/v) mixture of 12.5 mM NaPO₄ and acetonitrile. The glutamate peak occurs during the first 5 min after sample application when the mobile phase is isocratic phosphate buffer. The column was flushed by using a steep gradient (ending in 50% acetonitrile) before the next sample. This procedure allowed for the detection of glutamate at a sensitivity of 0.5 to 2.0 ng/sample. A dose-response curve was generated on each day of sampling. A PC-based data acquisition and analysis system (Millennium; Waters) was used to quantify glutamate based on the area under the curves.

Statistical Analysis. Behavioral data were analyzed for significance by analysis of variance for repeated measures followed by a post hoc Bonferroni analysis. Immunohistochemical data were analyzed for significance by one-way analysis of variance followed by a post hoc Bonferroni analysis. Glutamate and CGRP release data were analyzed by t test. P values <0.05 were considered significant. All statistical analysis was done with GraphPad Prism version 3.02 (GraphPad Software Inc., San Diego, CA; www.graphpad.com).

	Results

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Developmental Changes in PKC and PKC Expression. Expression of PKC expression in the L5 DRG increased with maturation. Approximately 41% of DRG cells expressed PKC in P7 rats. The number of PKC-positive cells within the DRG increased to 60% by P21 (Fig. 1, A–C; Table 1). At all three postnatal ages, PKC-immunoreactivity (-ir) was cytoplasmic by light microscopy. As previously reported, the largest diameter cell bodies, immunoreactive or not, are absent at P7 and P15 (Beland and Fitzgerald, 2001). At P21, PKC-ir was observed in both large- (big arrows) and small (small arrows)-diameter primary afferent cell bodies (Fig. 1, A–C).

View larger version (110K): [in this window] [in a new window]   Fig. 1. Expression of PKC in L5 dorsal root ganglion increased with postnatal maturation. In the absence of primary antibody, no appreciable staining is observed (A). Both small- and medium-diameter cell bodies showed PKC-ir in P7 (B) and P15 (C) rats. In P21 rats, PKC-ir was observed in both large- (large arrow), medium-, and small (small arrow)-diameter cell bodies (C). Scale bar, 100 µm.

View this table: [in this window] [in a new window]   TABLE 1 Age-specific PKC expression in L5 dorsal root ganglion neurons and PKC expression in lumbar spinal cord

The number of PKC-positive neuronal somata and the location of these cells within the spinal cord changed with age. Within the dorsal horn, PKC-positive cell somata increased in number and became localized to lamina II interneurons with postnatal age (Fig. 2, A–C; Table 1). In P7 rats, few cell somata stained positive for PKC in lamina II, although fiber-like PKC-ir within this lamina was observed (data not shown). In contrast, within the ventral horn, the number of PKC-positive neurons decreased with postnatal maturation (Fig. 2, D–F; Table 1). Ventral horn PKC-ir was predominantly nuclear in P7 rats. In P15 and P21 rats, PKC-ir was both nuclear and cytoplasmic in ventral horn motor neurons (Fig. 2, D–F).

View larger version (76K): [in this window] [in a new window]   Fig. 2. Age-specific expression of PKC in the lumbar spinal cord. In lamina II, the number of PKC-ir neuronal cell somata increased with postnatal maturation with few apparent at P7 (A) compared with P15 (B) or P21 (C). In contrast, PKC-ir in the ventral horn decreased with postnatal maturation with fewer PKC-ir motor neurons at P21 (F) compared with P15 (E) or P7 (D). Scale bar, 30 µm (A–C) and 100 µm (D–F).

Translocation of and PKC after Intraplantar Formalin and Prevention by Isozyme-Specific Inhibitors. Using densitometry, decreased PKC-ir in DRG was observed at 5 and 10 min postformalin at all three ages (Table 2; Fig. 3B). Intrathecal administration of the PKC translocation inhibitor V1-2 (50 pmol/5 µl) prevented the loss of PKC-ir after formalin (Table 2; Fig. 3C). At postnatal day 21, a decrease in PKC-ir was observed in both small- and large-diameter primary afferent cell bodies after formalin (Fig. 3B). Inhibition of PKC translocation with V3-5 did not alter PKC-ir at any age (data not shown). By Western analysis, intraplantar formalin did not alter PKC protein levels in DRGs from P7 and P21 rats (Fig. 3).

View this table: [in this window] [in a new window]   TABLE 2 Translocation of PKC in L5 dorsal root ganglion neurons from P7, P15, or P21 rats after intraplantar formalin or saline A loss of PKC-ir in DRG after intraplantar formalin compared with intraplantar saline or normal naïve tissue was indicative of translocation. Pretreatment with V1-2 (50 pmol/5 µl, intrathecal 15 min before formalin), but not Tat carrier, blocked the loss of PKC-ir in DRG indicative of translocation. Data represented as average ± S.D.

View larger version (112K): [in this window] [in a new window]   Fig. 3. Formalin produced PKC translocation in DRGs from P21 rats. A, DRGs had cytoplasmic PKC-ir 10 min postintraplantar saline. B, in contrast, a loss of cytoplasmic PKC-ir was seen at 10 min postformalin. This was blocked by V1-2 (C; 50 pmol/5 µl, intrathecal 15 min before formalin) but not Tat carrier (D; 50 pmol/5 µl, intrathecal 15 min before formalin). Scale bar, 100 µm. E, by Western analysis, no change in PKC expression in L4–L6 DRG was observed under the same conditions that decreased PKC-ir. Each band represents a separate sample from normal, 5 or 10 min postsaline, 5 or 10 min postformalin, or 10 min postformalin and pre-treated with intrathecal Tat, V1-2, or V5-3.

Intraplantar formalin did not alter PKC-ir in the ventral horn at any postnatal age (data not shown). No change in the intensity, number of positive staining cells or gross intracellular localization of PKC-ir within the dorsal horn was observed at 5 or 10 min postformalin in P7 and P15 rats. In contrast, increased PKC-ir in lamina II of the lumbar spinal cord was observed at 10 min postformalin in P21 rats (Fig. 4C; Table 3). The increase in PKC-ir was prevented by intrathecal administration of V3-5 (50 pmol/5 µl) (Fig. 4D). Interestingly, in P21 rats, V1-2 attenuated the formalin-induced increase in lamina II PKC-ir (Fig. 4E). By Western analysis, intraplantar formalin did not alter PKC protein levels in the lumbar spinal cord from P7 and P21 rats (Fig. 4G).

View larger version (96K): [in this window] [in a new window]   Fig. 4. Formalin produced translocation of PKC in lamina II of the lumbar spinal cord from P21 rats. Increased PKC-ir was observed in lamina II of the dorsal spinal cord at 10 min postformalin (C) compared with postsaline (B) or normal naive (A). The increase in PKC-ir was blocked by administration of V5-3 (D; 50 pmol/5 µl, intrathecal 15 min before formalin), V1-2 (E; 50 pmol/5 µl, intrathecal 15 min before formalin), but not Tat carrier peptide (F; 50 pmol/5 µl, intrathecal 15 min before formalin). Scale bar, 30 µm. G, by Western analysis, no change in PKC expression in the lumbar spinal cord was observed under the same conditions that increased PKC-ir. Each band represents a separate sample from normal, 5 or 10 min postsaline, 5 or 10 min postformalin, or 10 min postformalin and pretreated with intrathecal Tat, V1-2, or V5-3.

View this table: [in this window] [in a new window]   TABLE 3 Translocation of PKC in lamina II from L5 spinal cord from P7, P15, or P21 rats after intraplantar formalin An increase in PKC-ir in lumbar spinal cord after intraplantar formalin compared with intraplantar saline or normal naïve tissue was indicative of translocation. Pretreatment with V1-2 or V5-3 (50 pmol/5 µl, intrathecal 15 min before formalin), but not Tat carrier, blocked the change in PKC-ir in lumbar spinal cord. Data represented as average ± S.D.

Modulation of Formalin-Induced Nociception by PKC: Controls. Intraplantar saline produced mild and transient behaviors that were more marked in P7 (Fig. 5A) rats than in P15 and P21 rats (Fig. 5, B and C, respectively). Intrathecal administration of V1-2, V5-3, or Tat carrier 15 min before intraplantar saline did not alter saline-associated responses (Fig. 5, A–C). Tat carrier administration did not alter age-specific formalin responses compared with saline vehicle (Fig. 5, D–F).

View larger version (32K): [in this window] [in a new window]   Fig. 5. Isozyme-specific inhibitors of PKC translocation do not alter normal or saline-evoked behaviors. Intrathecal administration of V1-2, V5-3, or Tat carrier had no effect on normal responses in the 15 min immediately after administration or in behavioral responses after intraplantar saline in P7 (A), P15 (B), or P21 (C) rats (n = 6/group). Age-specific formalin responses were not altered by intrathecal Tat carrier compared with intrathecal saline in P7 (D), P15 (E), or P21 (F) rats (n = 6/group).

Modulation of Formalin-Induced Nociception by and PKC. Intrathecal administration of V1-2 dose dependently attenuated phase 1 and phase 2 behaviors in all ages (Fig. 6). Statistical analysis of V1-2 antinociception as a function of phase of the formalin response is shown in Fig. 6D. In P7 rats the age-specific monophasic formalin response was artificially split into three phases for behavioral comparison with older rats that display the stereotypic phase 1, quiescence and phase 2 formalin response. Furthermore, for all ages phase 2 was defined as beginning at 18 min and ending when behaviors significantly higher than baseline behaviors ceased. In P7 rats, 50 or 100 pmol/5 µl of V1-2 produced a biphasic behavioral profile and attenuated behaviors in all phases of the formalin response (Fig. 6A). In P15 rats, all doses of V1-2 attenuated phase 2 behaviors, whereas only the highest dose attenuated phase 1 behaviors (Fig. 6B). In P21 rats, all doses of V1-2 attenuated phase 2 behaviors, and all but the lowest dose attenuated phase 1 behaviors (Fig. 6C).

View larger version (47K): [in this window] [in a new window]   Fig. 6. Inhibition of PKC translocation with V1-2 dose-dependently and age-independently attenuated phase 1 and phase 2 formalin behaviors in P7 (A), P15 (B), and P21 (C) rats (n = 8–10/group). D, statistical analysis of V1-2 antinociception as a function of phase of the formalin response.

In contrast to PKC, inhibition of PKC translocation dose dependently attenuated only phase 2 formalin responses (Fig. 7). Statistical analysis of V3-5 antinociception as a function of phase of the formalin response is shown in Fig. 7D. Attenuation of phase 2 behaviors was age-dependent. In P7 rats, V3-5 did not alter formalin-induced pain behaviors at any dose examined (Fig. 7A). In P15 rats, the two higher doses of V3-5 attenuated phase 2 behaviors (Fig. 7B). In P21 rats, all doses of V3-5 examined attenuated phase 2 behaviors (Fig. 7C).

View larger version (46K): [in this window] [in a new window]   Fig. 7. Inhibition of PKC translocation with V5-3 dose-dependently attenuated phase 2 formalin behaviors inP15 (B) and P21 (C) rats but not in P7 rats (A; n = 8–10/group). D, statistical analysis of V3-5 antinociception as a function of phase of the formalin response.

Modulation of Neurotransmitter Release by PKC. Whole spinal cords with intact nerve roots were isolated from P7 rats and incubated in artificial cerebral spinal fluid. Maximal release of CGRP was elicited by 25 µM capsaicin (Table 4). Capsaicin-stimulated release of CGRP was Ca²⁺-dependent as shown by a loss of significant release in Ca²⁺-free artificial cerebrospinal fluid (Table 4). Bath application of the nonspecific PKC inhibitor GF109203X (1.2 µM), but not the calcium-dependent PKC antagonist Go6976 (1 µM), reduced CGRP release by 34% (Table 5). Bath application of V1-2 (25.6 pmol), but not Tat carrier (25.6 pmol), reduced glutamate release by 20% and CGRP release by 55% (Table 5). At the end of the release assays, electrophysiological recording of the nociceptive related slow ventral root potential (Feng and Kendig, 1996) in the isolated spinal cords showed the cords remained viable at the conclusion of the experiment (data not shown).

View this table: [in this window] [in a new window]   TABLE 5 Capsaicin (25 µ M)-stimulated release of glutamate and CGRP from spinal cords isolated from P7 rats was attenuated by V1-2 Data represented as average ± S.D. (n = 4/group).

	Discussion

Top Abstract Materials and Methods Results Discussion Conclusions References

 
Inhibition of PKC translocation in P7, P15, and P21 rats and PKC translocation in P15 and P21 rats attenuated formalin-induced nociception. The PKC inhibitor V1-2 attenuated phase 1 and phase 2 behaviors, whereas the PKC inhibitor V5-3 only attenuated phase 2 behaviors. These behavioral findings correlate well with the age-independent expression and translocation of PKC in DRG and the age-dependent expression and translocation of PKC in the lumbar spinal cord after formalin. Furthermore, the current study specifically identifies a role for PKC within primary afferents in capsaicin-stimulated release of glutamate and CGRP.

Developmental Regulation of and PKC Expression. Similar to findings reported by Beland and Fitzgerald (2001), there were few large-diameter cells in P7 DRG. This supports the postulate that differentiation of primary afferent subtypes occurs after the first postnatal week. Expression of PKC in the DRG is developmentally regulated with increasing expression across maturation. In the present study, large-diameter cells were observed in P21 rats, and many of these cells were PKC-ir. In adult rats, 90% of dorsal root ganglion cells express PKC (Khasar et al., 1999), suggesting that much of the maturation of PKC in primary afferents occurs during the second and third postnatal weeks.

Embryonic and postnatal PKC expression and activity differ between spinal cord and brain (Hashimoto et al., 1988; Akinori, 1998). Studies in the rodent brain suggest that PKC is expressed only postnatally and that kinase activity increases with maturation (Hashimoto et al., 1988). In contrast, PKC-ir is evident prenatally in neuronal somata in the dorsal and ventral horns of the spinal cord (Akinori, 1998). Similar to the findings by Akinori (1998), the present study reports a decrease in PKC-ir cells with age; by postnatal day 21, only large motor neurons and lamina II interneurons are PKC-ir. By adulthood, only 30% of ventral horn motor neurons show PKC-ir (Akinori, 1998), suggesting that like PKC, maturation of PKC in the lumbar spinal cord occurs during the first few postnatal weeks.

In P7 rats PKC-ir in lamina II of the dorsal horn was fiber-like with few PKC-ir neuronal somata. The fiber-like appearance of PKC-ir suggests an association of PKC with neurofilaments and thus may be in an immature form. Alternatively, this fibrillar location of PKC may mediate cellular functions that are distinct from those associated with responses to pain in older rats. It has been suggested that PKC may be involved in synaptogenesis and the development of postsynaptic functions (Akinori, 1998). This highlights the importance of developing and using PKC isozyme-specific inhibitors to avoid the confounding developmental compensation issues surrounding traditional gene knockout technology.

Translocation of and PKC after Formalin. Decreased PKC-ir in the dorsal root ganglion and increased PKC-ir in the dorsal horn of the spinal cord were observed after intraplantar formalin. We speculate that these two opposite observations both reflect translocation. A decline or an increase in immunoreactivity in fixed tissue is likely to reflect activation-dependent changes in the accessibility of the antibodies to the activated enzyme. The commercial antibodies are generated against a small antigenic determinant at the C terminus of the enzyme; when the enzyme is translocated and associated with its RACK (Johnson et al., 1996; Csukai et al., 1997), and/or when the antigenic site is phosphorylated (Keranen et al., 1995), binding of the antibodies can be altered. Furthermore, the decline in immunoreactivity was not due to degradation of the enzyme (as indicated by Western blot analysis of cell lysates), indicating that use-dependent degradation of the enzyme did not occur due to the noxious stimulus. Importantly, the decline in immunoreactivity of PKC was dependent upon its translocation; in the presence of the selective PKC translocation inhibitor V1-2 (Johnson et al., 1996; Nowak et al., 2003), the decline was inhibited. In contrast, in the presence of the Tat carrier, or the selective translocation inhibitor of PKC V5-3, the activation-induced decline in immunoreactivity was not altered. Similarly, increases in PKC-ir were selectively blocked by the PKC-specific inhibitor in the absence of protein expression changes by Western blot analysis.

Inhibition of Pain: PKC. Blocking PKC translocation attenuated both phase 1 and 2 of the formalin response in P7, P15, and P21 rats. The current findings suggest that activation of PKC in primary afferents precedes and possibly induces PKC activation in lamina II of the spinal cord. That activation of PKC as a primary event in phase 1, from which PKC activation and phase 2 behavioral responses result, is suggested by several findings. First, the anatomical location of these two distinct PKC isozymes supports the following cascade of events: activation of PKC in primary afferents, followed by PKC in dorsal horn interneurons. Second, inhibition of PKC attenuates both phase 1 and phase 2 pain behaviors, whereas inhibition of PKC only alters phase 2 behaviors. Furthermore, inhibition of PKC prevented translocation of both and PKC, whereas inhibition of PKC only modified PKC translocation. The independence of PKC actions from PKC signaling is further illustrated in P7 rats, which are sensitive to anti-PKC therapy but not to inhibition of PKC.

We have shown PKC-ir in small- and medium-diameter dorsal root ganglion cells, suggesting expression in nociceptors. In addition, we show that PKC mediates the release of the excitatory neurotransmitter glutamate and the neuro-modulatory transmitter CGRP in isolated spinal cords stimulated with capsaicin to activate VR1-expressing primary afferents. This suggests that PKC is also expressed in the central terminals of small- and medium-diameter dorsal root ganglion cells and involved in the release of the pronociceptive peptide CGRP. Furthermore, it has been reported that PKC may regulate depolarization and firing in dorsal root ganglion neurons through regulation of tetrodotoxin resistant sodium currents (Khasar et al., 1999). These findings expand on previous in vitro studies demonstrating enhanced presynaptic release of neuropeptides (Barber and Vasko, 1996; Frayer et al., 1999) and increased depolarization and firing of dorsal root ganglion neurons (Zhou et al., 2001) after nonspecific activation of PKC. Thus, we conclude that PKC, and specifically PKC, modulates release of excitatory neurotransmitters and neuromodulatory peptides from central terminals of primary afferents.

Inhibition of Pain: PKC. The present study provides direct evidence that selective inhibition of PKC in vivo attenuates phase 2 formalin-induced pain behaviors in an age-specific manner. These findings expand on earlier studies in adult rats showing that nonspecific inhibitors of PKC attenuate phase 2 formalin responses (Yashpal et al., 1995) and increase PKC immunoreactivity in lamina II after formalin (Malmberg et al., 1997) and that adult mice deficient in PKC have decreased formalin-induced spontaneous pain behaviors (Malmberg et al., 1997). In our study, inhibition of PKC translocation did not alter PKC-ir or formalin-induced spontaneous pain behaviors in P7 rats compared with P21 rats. It is possible that in P7 rats, formalin induces changes in PKC-ir at later times than were examined in this study. Alternatively, small changes in PKC-ir may not be detectable by light microscopy. Regardless of the limitations of the techniques used in the present study, there are clearly differences in the time course and magnitude of translocation and these differences are age-dependent. Furthermore, these findings suggest phase 2 formalin responses may be independent of PKC in P7 rats.

In P15 and P21 rats, central sensitization may be mediated presynaptically or postsynaptically on interneurons. PKC has been reported to increase the release of glutamate and aspartate in the spinal cord (Gerber et al., 1989). We have presented evidence in this study that PKC in primary afferents modulates the release of excitatory glutamate and CGRP, providing a mechanism for presynaptic modulation of spinal interneurons. Postsynaptically, PKC is expressed in excitatory interneurons in lamina II of the dorsal horn (Polgar et al., 1999), and PKC has been shown to alter N-methyl-D-aspartate-induced currents in the spinal cord (Chen and Huang, 1992; Martin et al., 2001). More recently, it has been reported that N-methyl-D-aspartate antagonism blocks formalin induced PKC translocation in the superficial dorsal horn (Chen and Huang, 1992; Yashpal et al., 2001) further supporting presynaptic modulation of postsynaptic neurons containing PKC.

	Conclusions

Top Abstract Materials and Methods Results Discussion Conclusions References

 
Very young infants experience both noxious and some innocuous stimuli as a physiologically disturbing event (Fitzgerald et al., 1989; Fitzgerald and Anand, 1993). Furthermore, early painful experiences may predispose infants and children to a later enhancement in both physiological and cognitive/emotional responses to noxious insult (Taddio et al., 1997; Alvares et al., 2000; Ruda et al., 2000; Lidow et al., 2001). Thus, it is imperative to understand age-specific regulation of pain pathways for the rational development of nonopioid analgesics for managing pain in infants and children. The present study provides evidence that pharmacological manipulation of selective PKC isozymes may offer several attractive therapeutic targets for the management of pain in pediatrics and that therapies will need to be tailored to the different roles played by PKC isozymes at different ages.

	Footnotes

 
This work was supported by National Institute of Health Grants DA08256 (to D.C.Y.), NS13108 (to J.J.K.), NS4472901 (to S.M.S.), and the Alejandro and Lida Zaffaroni Innovation Fund for Addiction Research (to S.M.S.).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

DOI: 10.1124/jpet.103.060350.

ABBREVIATIONS: P, postnatal day; PKC, protein kinase C; DAG, diacylglycerol; RACK, receptors for activated C kinase; DRG, dorsal root ganglia; PBS, phosphate-buffered saline; CGRP, calcitonin gene-related peptide; ir, immunoreactivity; Go6976, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole; GF109203X, bisindolylmaleimide I hydrochloride.

Address correspondence to: Dr. Sarah M. Sweitzer, Department of Pharmacology, Physiology, and Neuroscience, University of South Carolina School of Medicine, Columbia, SC 29208. E-mail: sweitzer{at}gw.med.sc.edu

	References

Top Abstract Materials and Methods Results Discussion Conclusions References

 

Akinori M (1998) Subspecies of protein kinase C in the rat spinal cord. Prog Neurobiol 54: 499-530.[Medline]
Alvares D, Torsney C, Beland B, Reynolds M, and Fitzgerald M (2000) Modelling the prolonged effects of neonatal pain. Prog Brain Res 129: 365-373.[Medline]
Barber LA and Vasko MR (1996) Activation of protein kinase C augments peptide release from rat sensory neurons. J Neurochem 67: 72-80.[Medline]
Beland B and Fitzgerald M (2001) Mu- and delta-opioid receptors are downregulated in the largest diameter primary sensory neurons during postnatal development in rats. Pain 90: 143-150.[CrossRef][Medline]
Chen L and Huang L (1992) Protein kinase C reduces Mg2+ block of NMDA-receptor channels as a mechanism of modulation. Nature (Lond) 356: 521-523.[CrossRef][Medline]
Chen L, Wright LR, Chen CH, Oliver SF, Wender PA, and Mochly-Rosen D (2001) Molecular transporters for peptides: delivery of a cardioprotective epsilonPKC agonist peptide into cells and intact ischemic heart using a transport system, R(7). Chem Biol 8: 1123-1129.[CrossRef][Medline]
Coderre TJ, Vaccarino AL, and Melzack R (1990) Central nervous system plasticity in the tonic pain response to subcutaneous formalin injection. Brain Res 535: 155-158.[CrossRef][Medline]
Csukai M, Chen CH, De Matteis MA, and Mochly-Rosen D (1997) The coatomer protein 'COP: a selective binding protein (RACK) for epsilon protein kinase C. J Biol Chem 272: 29200-29206.[Abstract/Free Full Text]
Dickenson AH and Sullivan AF (1987) Peripheral origins and central modulation of subcutaneous formalin-induced activity of rat dorsal horn neurones. Neurosci Lett 83: 207-211.[CrossRef][Medline]
Feng J and Kendig JJ (1996) The NMDA receptor antagonist MK-801 differentially modulates mu and kappa opioid actions in spinal cord in vitro. Pain 66: 343-349.[CrossRef][Medline]
Fitzgerald M and Anand K (1993) The developmental neuroanatomy and neurophysiology of pain, in Pain Management in Infants, Children and Adolescents (Schetchter NL, Berde CB, and Yaster M eds) pp 11-32, Lippincott Williams & Wilkins, Baltimore, MD.
Fitzgerald M, Catherine M, and McIntosh N (1989) Cutaneous hypersensitivity following peripheral tissue damage in newborn infants and its reversal with topical anaesthesia. Pain 39: 31-36.[CrossRef][Medline]
Frayer SM, Barber LA, and Vasko MR (1999) Activation of protein kinase C enhances peptide release from rat spinal cord slices. Neurosci Lett 265: 17-20.[CrossRef][Medline]
Gerber G, Kangrga I, Ryu P, Larew J, and Randic M (1989) Multiple effects of phorbol esters in the rat spinal dorsal horn. J Neurosci 9: 3606-3617.[Abstract]
Graser TA, Godel HG, Albers S, Foldi P, and Furst P (1985) An ultra rapid and sensitive high-performance liquid chromatographic method for determination of tissue and plasma free amino acids. Anal Biochem 151: 142-152.[CrossRef][Medline]
Hashimoto T, Ase K, Sawamura S, Kikkawa U, Saito N, Tanaka C, and Nishizuka Y (1988) Postnatal development of brain-specific subspecies of protein kinase C in rat. J Neurosci 8: 1678-1683.[Abstract]
Johnson JA, Gray MO, Chen CH, and Mochly-Rosen D (1996) A protein kinase C translocation inhibitor as an isozyme-selective antagonist of cardiac function. J Biol Chem 271: 24962-24966.[Abstract/Free Full Text]
Keranen LM, Dutil EM, and Newton AC (1995) Protein kinase C is regulated in vivo by three functionally distinct phosphorylations. Curr Biol 5: 1394-1403.[CrossRef][Medline]
Khasar SG, Lin YH, Martin A, Dadgar J, McMahon T, Wang D, Hundle B, Aley KO, Isenberg W, McCarter G, et al. (1999) A novel nociceptor signaling pathway revealed in protein kinase C epsilon mutant mice. Neuron 24: 253-260.[CrossRef][Medline]
Lidow M, Song Z, and Ren K (2001) Long-term effects of short-lasting early local inflammatory insult. Neuroreport 12: 399-403.[Medline]
Malmberg AB, Chen C, Tonegawa S, and Basbaum AI (1997) Preserved acute pain and reduced neuropathic pain in mice lacking PKCgamma. Science (Wash DC) 278: 279-283.[Abstract/Free Full Text]
Marsh D, Dickenson A, Hatch D, and Fitzgerald M (1999) Epidural opioid analgesia in infant rats I: mechanical and heat responses. Pain 82: 23-32.[CrossRef][Medline]
Martin WJ, Malmberg AB, and Basbaum AI (2001) PKCgamma contributes to a subset of the NMDA-dependent spinal circuits that underlie injury-induced persistent pain. J Neurosci 21: 5321-5327.[Abstract/Free Full Text]
Mochly-Rosen D and Gordon A (1998) Anchoring proteins for protein kinase C: a means for isozyme selectivity. FASEB J 12: 35-42.[Abstract/Free Full Text]
Nowak G, Bakajsova D, and Clifton G (2004) Protein kinase C modulates mitochondrial function and active Na+ transport following oxidant injury in renal cells. Am J Physiol Renal Physiol 286: 307-316.
Polgar E, Fowler JH, McGill MM, and Todd AJ (1999) The types of neuron which contain protein kinase C gamma in rat spinal cord. Brain Res 833: 71-80.[CrossRef][Medline]
Puig S and Sorkin LS (1996) Formalin-evoked activity in identified primary afferent fibers: systemic lidocaine suppresses phase-2 activity. Pain 64: 345-355.[CrossRef][Medline]
Ruda M, Qing-Dong L, Hohmann A, Peng Y, and Toshiya T (2000) Altered nociceptive neuronal circuits after neonatal peripheral inflammation. Science (Wash DC) 289: 628-630.[Abstract/Free Full Text]
Skilling SR, Smullin DH, Beitz AJ, and Larson AA (1988) Extracellular amino acid concentrations in the dorsal spinal cord of freely moving rats following veratridine and nociceptive stimulation. J Neurochem 51: 127-132.[Medline]
Sweitzer S, Colburn R, Rutkowski M, and DeLeo J (1999) Acute peripheral inflammation induces moderate glial activation and spinal interleukin-1 beta expression that correlates with pain behavior in the rat. Brain Res 829: 209-221.[CrossRef][Medline]
Sweitzer S, Wong S, Tjolsen A, Allen C, Mochly-Rosen D, and Kendig JJ (2004) Exaggerated nociceptive responses on morphine withdrawal: roles of protein kinase C epsilon and gamma. Pain, in press.
Taddio A, Katz J, Ilersich AL, and Koren G (1997) Effect of neonatal circumcision on pain response during subsequent routine vaccination. Lancet 349: 599-603.[CrossRef][Medline]
Taylor BK, Peterson MA, and Basbaum AI (1995) Persistent cardiovascular and behavioral nociceptive responses to subcutaneous formalin require peripheral nerve input. J Neurosci 15: 7575-7584.[Abstract]
Teng C and Abbott F (1998) The formalin test: a dose-response analysis at three developmental stages. Pain 76: 337-347.[CrossRef][Medline]
Yashpal K, Fisher K, Chabot JG, and Coderre TJ (2001) Differential effects of NMDA and group I mGluR antagonists on both nociception and spinal cord protein kinase C translocation in the formalin test and a model of neuropathic pain in rats. Pain 94: 17-29.[CrossRef][Medline]
Yashpal K, Pitcher G, Parent A, Quirion R, and Coderre T (1995) Noxious thermal and chemical stimulation induce increases in 3H-phorbol 12,13-dibutyrate binding in spinal cord dorsal horn as well as persistent pain and hyperalgesia, which is reduced by inhibition of protein kinase C. J Neurosci 15: 3263-3272.[Abstract]
Zhou Y, Zhou ZS, and Zhao ZQ (2001) PKC regulates capsaicin-induced currents of dorsal root ganglion neurons in rats. Neuropharmacology 41: 601-608.[CrossRef][Medline]

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