Introduction

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Isoprostanes are stereoisomers of prostaglandins that are formed primarily through the nonenzymatic in situ peroxidation of arachidonic acid by reactive oxygen species (Morrow et al., 1990, 1994). Although a large number of these isoprostanes have been identified (see reviews by Roberts and Morrow, 1997; Rokach et al., 1997), studies on the biological activity of isoprostanes have focused primarily on 8-iso prostaglandin F₂ (8-iso PGF₂) and 8-iso prostaglandin E₂ (8-iso PGE₂). Both of these isoprostanes are potent vasoconstrictors (Morrow et al., 1990, 1994), and both affect platelet aggregation (Longmire et al., 1994; Pratico et al., 1996). In addition, 8-iso PGF₂ appears to be a useful indicator of lipid peroxidation because micromolar concentrations of this isoprostane are generated during injury associated with oxidative stress (Reilly et al., 1999).

Although isoprostanes have potent actions on the vascular system, the question remains whether these novel eicosanoids affect nociceptive sensory neurons in a manner analogous to prostaglandins. Indeed, it is well established that proinflammatory prostaglandins, such as PGE₂ and PGI₂, produce hyperalgesia (Ferreira et al., 1978), in part, from their ability to lower the firing threshold of nociceptive sensory neurons (Handwerker, 1976; Schaible and Schmidt, 1988; Nicol and Cui, 1994) and to augment the release of transmitters from sensory nerve terminals (Franco-Cereceda, 1989; Hingtgen and Vasko, 1994; Vasko et al., 1994). If isoprostanes cause hyperalgesia and alter the sensitivity of sensory neurons, then they could represent a novel class of algogens whose mechanism of action is analogous to prostaglandins. Furthermore, the actions of isoprostanes on pain might not be attenuated by nonsteroidal anti-inflammatory drugs (NSAIDs), because a major pathway leading to the formation of these unique prostanoids is independent of cyclooxygenases (Morrow et al., 1992; Pratico et al., 1995).

To assess the potential role of isoprostanes as proinflammatory mediators involved in nociception, we examined whether isoprostanes enhanced thermal or mechanical nociception in animal models of pain and whether these agents modified the electrophysiological properties of C-nociceptive fibers in pentobarbital-anesthetized rats. To further ascertain the effects of isoprostanes on sensory neurons at a cellular level, we examined whether these eicosanoids altered the excitability of, and enhanced the release of, transmitters from isolated sensory neurons grown in culture. Our findings demonstrate that 8-iso PGE₂ produces nociception and sensitizes sensory neurons, suggesting that this isoprostane may be an important substance in mediating pain through a direct action on sensory neurons. Portions of this work appeared previously in abstract form (Evans et al., 1997; Junger et al., 1998).

	Experimental Procedures

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Materials. Timed-pregnant and male Sprague-Dawley rats and male Holtzmann rats were purchased from Harlan Sprague-Dawley, Inc. (Indianapolis, IN). Cell culture supplies were purchased from Life Technologies Bethesda Research Labs (Grand Island, NY) and nerve growth factor from Harlan Bioproducts for Science, Inc. (Indianapolis, IN). The substance P (SP) antiserum was raised in the laboratory of Dr. Vasko, and the calcitonin gene-related peptide (CGRP) antiserum was donated by Dr. Michael Iadorola (National Institutes of Health). Isoprostanes were purchased from Caymen Chemical Co. (Ann Arbor, MI); SP, CGRP, and bradykinin from Peninsula Laboratories (Belmont, CA); ketorolac from Syntax Laboratories (Palo Alto, CA); and other chemicals from Sigma Chemical Co. (St. Louis, MO). 8-Iso PGE₂, 8-iso PGF₂, and capsaicin were dissolved initially in 1-methyl-2-pyrrolidinone (Aldrich Chemical Co., Milwaukee, WI) and then diluted to the appropriate concentrations with sterile saline or buffers (see below) to yield the appropriate concentration. We have demonstrated previously that the vehicle, 1-methyl-2-pyrrolidinone, had no effect on the excitability of or transmitter release from sensory neurons (Nicol and Cui, 1994; Vasko et al., 1994). All procedures were approved by the Animal Care and Use Committees at Indiana University School of Medicine (Indianapolis, IN) and at the University of California, San Diego (La Jolla, CA).

Behavioral Nociceptive Assessment. Male Sprague-Dawley rats (300-350 g) were housed two per cage and maintained on a 12-h dark/light cycle with food and water ad libitum. To assess mechanical withdrawal thresholds, each animal was placed in a Plexiglas cage (25.5 cm × 10 cm × 15 cm) with a wire mesh bottom supported by a polyvinylchloride framework (60 cm in height). Thresholds were measured using eight von Frey hairs with buckling forces of 4.0, 6.8, 11.8, 20.0, 35.6, 53.9, 83.4, and 148 mN (Stoelting, Wood Dale, IL). Beginning with the 20-mN hair, each von Frey hair was presented perpendicular to the plantar surface of the hindpaw for 6 s with sufficient force to cause slight buckling (Chaplan et al., 1994). Hairs were presented in ascending order of stiffness until paw withdrawal was noted or the stiffest (148-mN) hair was applied. When a response was observed, hairs of decreasing stiffness were applied until the animal did not withdraw, at which point hairs were presented again in ascending order. This pattern was repeated for four stimulus presentations after the first withdrawal response. Both hindpaws were examined at each time point; the first paw (left or right) tested was alternated. Mechanical withdrawal thresholds were then calculated using the "up-down" method of Dixon (Chaplan et al., 1994).

Single Unit Recordings. The preparation and procedures used in the single unit recordings have been described in detail previously (Puig and Sorkin, 1995). Briefly, Sprague-Dawley or Holtzman rats (male, 300-400 g; Harlan Industries, Indianapolis, IN) were anesthetized with an i.p. injection of 50 mg/kg pentobarbital sodium. The sural nerve was exposed and the foot positioned on a clay block to prevent movement during application of mechanical stimuli. The sural nerve was separated from adjacent tissue and cut from the sciatic nerve. The skin was attached to a metal frame, which was used to form a pool of mineral oil that prevented the exposed tissue from drying and cooling. A silver reference electrode was inserted between the skin and the underlying muscle. About 1.5 to 2.0 cm distal from the cut end of the nerve, two silver wire electrodes were positioned to provide stimuli. On removal of the epineuria and perineuria, the cut end of the nerve was dissected into small fiber bundles. Neural activity was recorded using a silver hook electrode placed under the dissected fiber bundle.

Isolation and Culture of Embryonic Rat Sensory Neurons. The procedures for isolation and culture of rat sensory neurons are modified from those described previously (Vasko et al., 1994). Briefly, timed-pregnant rats were rendered unconscious with CO₂ and sacrificed by cervical dislocation. Embryos (E15-E17) were removed from the uterus and placed in a dish containing calcium-free and magnesium-free Hanks' balanced salt solution. The dorsal root ganglia were dissected from each embryo and sensory neurons were dissociated from the ganglia with 0.025% trypsin (37°C, 25 min) and mechanical agitation. Approximately 150,000 cells/well were plated on collagen-coated 24-well culture dishes. Neurons were grown in Dulbecco's modified Eagle's medium supplemented with 2 mM glutamine, 50 µg/ml penicillin and streptomycin, 10% (v/v) heat-inactivated fetal bovine serum, 50 µM 5-fluoro-2'-deoxyuridine, 150 µM uridine, and 250 ng/ml 7S nerve growth factor. Cultures were maintained at 37°C in a 5% CO₂, 95% air atmosphere. The medium was changed every second day.

Whole-Cell Patch-Clamp Recordings. Current-clamp recordings were made using the patch-clamp technique (Hamill et al., 1981). Briefly, a cover slip with the sensory neurons (grown 4-6 days in culture) was placed in a recording chamber and bathed in normal Ringer's solution of the following composition: 140 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, and 10 mM glucose, and adjusted to pH at 7.4 with NaOH. Recording pipettes were pulled from disposable borosilicate glass tubing and filled with the following solution: 140 mM KCl, 5 mM MgCl₂, 4 mM ATP, 0.3 mM GTP, 2.5 mM CaCl₂, 5 mM EGTA (calculated free calcium concentration of ~100 nM), and 10 mM HEPES, and adjusted to pH 7.3 with KOH. These pipettes typically had resistances of 2 to 5 M. Whole-cell voltages were recorded from sensory neurons using an EPC-7 patch-clamp amplifier (List Electronic, Darmstadt, Germany); the data were acquired and analyzed using pCLAMP6 (Axon Instruments, Foster City, CA). Ramps of depolarizing current of variable amplitude (50-900 pA) were applied to the neuron for 1 s. The amplitude was adjusted to produce one to three action potentials in any given neuron under control conditions. After obtaining the control response, the superfusate was changed to Ringer's containing isoprostanes, and cells were superfused continuously for various intervals. All experiments were performed at room temperature (~23°C). Perfusion with vehicle alone has been shown previously to have no effect on the number of action potentials elicited by the ramp of depolarizing current (Nicol and Cui, 1994).

Neuropeptide Release Protocol. Release experiments were performed on neurons after 9 to 12 days in culture. The cells were washed with 0.4 ml of HEPES buffer consisting of 25 mM HEPES, 135 mM NaCl, 3.5 mM KCl, 2.5 mM CaCl₂, 1 mM MgCl₂, 3.3 mM dextrose, 0.1 mM ascorbic acid, 0.02 mM bacitracin, 0.001 mM phosphorhamadon, and 0.1% BSA, pH 7.4, and maintained at 37°C. The release protocol consisted of three successive 10-min incubation periods. For those experiments examining the direct actions of isoprostanes on release, the three incubations consisted of a basal release (HEPES buffer alone), a stimulation period (HEPES buffer plus various concentrations of either 8-iso PGE₂ or 8-iso PGF₂), and another basal incubation period. In those experiments designed to determine whether isoprostanes augment evoked release, cells were exposed to either HEPES buffer or HEPES containing different concentrations of isoprostanes for an initial 10-min period. Next, the cultures were exposed for 10 min to buffer containing either 30 nM capsaicin or 100 nM bradykinin in the absence or presence of isoprostane to assess effects of the eicosanoids on evoked release. After this 10-min stimulation period, basal release was re-established by exposing the cells to HEPES buffer for 10 min. After each incubation, the buffer was removed from the culture wells, aliquoted, and the amounts of immunoreactive substance P (iSP) and immunoreactive calcitonin gene-related peptide (iCGRP) were measured using radioimmunoassay as previously described (Vasko et al., 1994). Using these two release protocols, we can assess whether the eicosanoids could alter resting release and whether it could augment stimulated release.

Data Analysis. In all instances except studies of mechanical allodynia, values represent the mean ± S.E. For the single unit recordings and thermal nociceptive behavioral studies, significant differences between baseline and drug-evoked thresholds were calculated with a one-way ANOVA and Scheffe F test for the post hoc analysis for repeated measures. Significant differences between groups were determined with Student's t test. For behavioral testing using von Frey hairs, significant differences between vehicle- and isoprostane-evoked thresholds were calculated using Friedman's test for repeated measures, then the Mann-Whitney U test for the post hoc test. For the current clamp recordings and for release studies, statistical differences between the controls and isoprostane treatments were determined by using an ANOVA with repeated measures (where appropriate). When a significant difference was obtained, post hoc analyses were performed using either a Student-Newman-Keul's or Fisher's least significant difference (LSD). Values of P < .05 were considered to be statistically significant.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Effects of Isoprostanes on Behavioral Nociception. To assess whether isoprostanes alter nociception, withdrawal thresholds or latencies were measured using mechanical or thermal stimuli, applied to the plantar surface of the hindpaw before and at various intervals after the intradermal injection (2.5 µl) of vehicle, 1.14 mM (1 µg) 8-iso PGE₂, or 1.14 mM (1 µg) 8-iso PGF₂. Injection of vehicle did not significantly reduce the mechanical withdrawal threshold over 120 min of testing (Fig. 1, top). We set the upper limit for testing at 148 mN to prevent misinterpretation of results (i.e., the possibility that the von Frey hair lifts the paw). As such, the data at 148 mN likely represent an underestimate of the mechanical threshold in vehicle-treated animals. For example, after injection of vehicle in 10 animals, 27 withdrawal measurements at different time points were 148 mN, 22 showed no response, and 5 responded to the 148-mN hair the first but not the second time the paw was tested. In contrast, 15 min after intradermal administration, 8-iso PGE₂ caused a significant reduction in withdrawal threshold compared with baseline values (n = 9, Fig. 1, middle). This action of 8-iso PGE₂ was maintained for 45 min and returned to control values by 60 min. In addition, rats injected with 8-iso PGE₂ responded to the mechanical stimulation by extensive licking of the injected paw after stimulation. Injection of 8-iso PGF₂ also produced a significant reduction in paw withdrawal threshold (n = 8, Fig. 1, bottom), but this effect was more transient than the sensitization caused by 8-iso PGE₂ because the withdrawal threshold returned to control values within 30 min after injection. There was no significant alteration in the withdrawal threshold in the contralateral paws after injection of either isoprostane or vehicle (data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Intradermal injection of 8-isoprostanes significantly reduces mechanical paw withdrawal threshold in rats. The ordinate represents paw withdrawal threshold in millinewtons, whereas the abscissa is the time in minutes after the intradermal injection of drug or vehicle. The wider horizontal lines under each time point (usually in the box) represent the median mechanical threshold for paw withdrawal using von Frey hairs for 8 to 10 rats. The top of the box is the 75th percentile, whereas the bottom of the box is the 25th percentile. Error bars above and below the box represent the 90th and 10th percentiles, respectively. Top, box plot of data from animals that received an intradermal injection of 2.5 µl of vehicle. Middle and bottom, mechanical threshold from rats receiving 1.14 mM (1 µg/2.5 µl) 8-iso PGE2 (n = 9) or 1.14 mM (1 µg/2.5 µl) 8-iso PGF2 (n = 8), respectively. An asterisk indicates significant difference from baseline using Friedman's test with the Mann-Whitney U test used for the post hoc analysis.

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View larger version (21K): [in this window] [in a new window]   Fig. 2.   Intradermal injection of 8-iso PGE2 augments thermal nociception. The ordinate represents thermal paw withdrawal latency in seconds, whereas the abscissa is the time in minutes after the intradermal injection of drug or vehicle. Each point shows the mean ± S.E. of the thermal latency for paw withdrawal for nine rats.  and  represent data from animals that received an intradermal injection of 1.14 mM (1 µg/2.5 µl) 8-iso PGE2 or 1.14 mM (1 µg/2.5 µl) 8-iso PGF2, respectively.  are thresholds from rats that were injected with 2.5 µl of vehicle. An asterisk indicates significant difference from vehicle using ANOVA with the Scheffe F test for the post hoc analysis.

Isoprostane Actions on Cutaneous Afferent Fibers. To ascertain whether isoprostanes altered the threshold for sustained firing of sensory neurons, the effects of these compounds were assessed after injection into the receptive fields of 121 nociceptive C fibers located predominantly in the glabrous portion of the sural innervation area. Nerves with receptive fields in the proximal heel area were excluded from study because the positioning of the foot during recording rendered full characterization of those fibers impractical. The conduction velocity for the C fibers was 0.77 ± 0.03 m/s. The force required to produce a sustained electrophysiological response (i.e., SRT) was 404 ± 50 mN. These afferents were also characterized into high-threshold mechanoreceptors (64.5% of the total), units responding to mechanical, heat, and cold stimulation (15.7%), mechano-heat units (16.5%), or mechano-cold units (3.3%). Mean thresholds for sustained firing were significantly higher in pure mechanoreceptors (498 ± 72 mN) than in C fibers with multiple modalities (283 ± 75 mN for units responding to mechanical, heat, and cold stimulation, 186 ± 50 mN for mechano-heat units, and 257 ± 165 mN for mechano-cold units). Mechanically insensitive C fibers were not investigated.

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View larger version (24K): [in this window] [in a new window]   Fig. 3.   Time course of sustained mechanical response thresholds in C fibers after s.c. injection of 8-iso PGE2 (top) or 8-iso PGF2 (bottom). Mechanical thresholds are expressed as percentage from baseline thresholds assessed before drug injection. Each point represents the mean ± S.E. for 7 to 27 fibers. Note that s.c. injection of 8-iso PGE2 resulted in a significant reduction in threshold at all concentrations except 1.14 µM. For 8-iso PGF2, concentrations of 114 and 1140 µM induced decreases of mechanical thresholds. An asterisk indicates significant difference from vehicle controls using ANOVA with the Scheffe F test for the post hoc analysis.

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Isoprostanes Enhance the Generation of Action Potentials in Isolated Sensory Neurons. Because a primary target mediating the nociceptive actions of prostaglandins is sensory neurons (Treede et al., 1992), we assessed whether isoprostanes altered the excitability of sensory neurons grown in culture. Isolated sensory neurons were current-clamped and a ramp of depolarizing current used to generate action potentials. As seen in Fig. 4A in a representative cell, the ramp of current elicited three action potentials under control conditions, whereas after a 10-min exposure to 1 µM 8-iso PGE₂ the same ramp now evoked nine action potentials (right). In 9 of 11 sensory neurons we examined, exposure to 8-iso PGE₂ for 2 min resulted in a significant increase in the number of action potentials. The ramp evoked 2.5 ± 0.4 action potentials (n = 11) under control conditions and 6.2 ± 1.0 action potentials 10 min after exposure to 8-iso PGE₂ (Fig. 4B). Exposing sensory neurons to 1 µM 8-iso PGF₂ also sensitized sensory neurons, although the number of action potentials elicited by the ramp of depolarizing current was augmented in only 6 of 13 cells examined. As summarized in Fig. 4B, 1 µM 8-iso PGF₂ increased the number of action potentials from 2.8 ± 0.3 (n = 13) under control conditions to 5.6 ± 0.9 after a 10-min treatment. Although the number of action potentials elicited by a depolarizing current was enhanced after isoprostane treatment, neither 8-iso PGE₂ nor 8-iso PGF₂ altered the resting membrane potential of the neurons. After a 20-min exposure to either isoprostane (1 µM), the resting membrane potential was 52 ± 2 mV for cells exposed to 8-iso PGE₂ (n = 11) or 55 ± 2 mV for cells treated with 8-iso PGF₂ (n = 13), compared with a value of 54 ± 1 mV (n = 24) before isoprostane treatment.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Isoprostanes augment the excitability of sensory neurons grown in culture. A, panels represent whole-cell patch-clamp recordings from a representative sensory neuron in current-clamp mode. Action potentials are generated in response to a ramp of injected current as indicated. The recording on the left is from a representative cell bathed in control buffer. On the right is the response of the same cell when exposed to 1 µM 8-iso PGE2 for 10 min. B, each column represents the mean ± S.E. of the number of action potentials elicited with a ramp of depolarizing current. Filled columns are action potentials generated by the ramp of depolarizing current in neurons exposed to 1 µM 8-iso PGE2 (n = 11), whereas open columns represent cells exposed to 1 µM 8-iso PGF2 (n = 13) for the times indicated. Columns on the left (C) indicate action potentials elicited before exposure to isoprostanes. Asterisks indicate significant differences between control and 8-isoprostane exposure using ANOVA and Student-Newman-Keul's test for the post hoc test.

Effects of Isoprostanes on Peptide Release. Based on the results presented above, isoprostanes, especially 8-iso PGE₂, augment the sensitivity of sensory neurons and enhance nociception. One possible mechanism to account for isoprostane-induced hyperalgesia is that these eicosanoids facilitate the release of transmitters from small-diameter sensory neurons. To examine this notion, we studied the effects of 8-iso PGE₂ or 8-iso PGF₂ on the release of iSP and iCGRP from rat sensory neurons grown in culture. As seen in Fig. 5, exposing isolated sensory neurons to 10 µM 8-iso PGE₂ for 10 min caused an approximately 3-fold increase in the basal release of iSP (n = 17). In a similar manner, the release of iCGRP was elevated 2-fold in cells exposed to 10 µM 8-iso PGE₂, a significant increase compared with release from cells exposed to buffer alone (n = 17; Fig. 5). Neither 0.1 nor 1.0 µM 8-iso PGE₂ or any of the concentrations of 8-iso PGF₂ tested had significant effect on basal peptide release.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Effects of isoprostanes on resting release of iSP and iCGRP from rat sensory neurons in culture. Each column represents the mean ± S.E. of iSP release (top) or iCGRP release (bottom) in femtomoles per well of cells for a 10-min incubation for the number of wells indicated. Open columns on the left represent basal (resting) release when cells are exposed to HEPES buffer without isoprostanes. Remaining open columns represent release from cells exposed to various concentrations of 8-iso PGF2 for 10 min, whereas filled columns show release in the presence of various concentrations of 8-iso PGE2. An asterisk denotes a significant difference from basal release in the absence of drug using ANOVA with Fisher's LSD for the post hoc test.

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View larger version (19K): [in this window] [in a new window]   Fig. 6.   Treatment with 8-iso PGE2 augments the bradykinin-evoked release of iSP and iCGRP. The ordinate represents amount of iSP (top) or iCGRP (bottom) released in femtomoles per well per 10-min incubation. Each square represents the mean ± S.E. of peptide release from 10 to 26 wells of sensory neurons from a minimum of three independent harvests.  indicate release from cultures examining the effects of various concentrations of 8-iso PGE2 and  represent studies using 8-iso PGF2. Points on the left (Basal) indicate the amount of peptide release when sensory neurons were exposed to HEPES buffer alone for 10 min. Adjacent to the basal release, the squares indicate the amounts of peptide released during a 10-min exposure of cells to 100 nM bradykinin. The concentration-response curves demonstrate the effects of exposing cells to various amounts of 8-iso PGE2 () or 8-iso PGF2 () in the presence of 100 nM bradykinin. Asterisks indicate significant differences between bradykinin-evoked release in the absence versus presence of isoprostanes using ANOVA with Fisher's LSD for the post hoc test.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Ketorolac pretreatment does not attenuate the 8-iso PGE2-induced augmentation of capsaicin-evoked release of iSP. Each column represents the mean ± S.E. of iSP release in femtomoles per well of cells for a 10-min incubation for 11 wells from three independent harvests of cells. Open columns represent basal or resting release when cells are exposed to HEPES buffer without capsaicin or 8-iso PGE2, whereas hatched columns show release in the presence of 1 µM 8-iso PGE2. Filled shaded columns represent release in the presence of 30 nM capsaicin (left) or in the presence of 30 nM capsaicin and 1 µM 8-iso PGE2 (right). Data in the top panel were from cells that were not pretreated with ketorolac, whereas the bottom panels represent release from cells pre-exposed to 100 nM ketorolac from 30 min before and throughout the 10-min basal and the 10-min stimulation periods as indicated. An asterisk denotes a significant difference from basal release using ANOVA and Newman-Keul's for the post hoc test, whereas a cross indicates a significant difference in capsaicin-evoked release in the presence of 1 µM 8-iso PGE2 compared with release with capsaicin alone.

Isoprostane-Induced Augmentation of Peptide Release Is Not Dependent on Prostaglandin Formation. Because PGE₂ and PGI₂ enhance the evoked release of iSP and iCGRP from sensory neurons (Hingtgen and Vasko, 1994; Vasko et al., 1994), we assessed whether the sensitizing action of 8-iso PGE₂ on peptide release could be secondary to prostaglandin synthesis via the cyclooxygenase pathway. For these studies, we examined the effects of this isoprostane in the presence of the cyclooxygenase inhibitor, ketorolac, on basal and capsaicin-evoked peptide release. Capsaicin was used as the stimulating agent rather than bradykinin because we have previously shown that treatment with NSAIDs attenuated bradykinin-stimulated release of iSP and iCGRP (Vasko et al., 1994). Exposing sensory neurons in culture to 30 nM capsaicin significantly increased the release of iSP by approximately 10-fold (Fig. 7, top). As with bradykinin-evoked release, pretreating cells with 1 µM 8-iso PGE₂ for 10 min before and throughout the capsaicin exposure caused a significant augmentation of the capsaicin-evoked peptide release (Fig. 7, top). Additional cultures from the same harvests were incubated with 100 nM ketorolac for 30 min before release studies. This concentration of ketorolac is 2- to 3-fold greater than the IC₅₀ for inhibition of cyclooxygenase (Riendeau et al., 1997). As can be seen in the bottom panel of Fig. 7, the cyclooxygenase inhibitor did not alter either the capsaicin-induced release of iSP or the sensitizing actions of 8-iso PGE₂. Similar results were observed with release of iCGRP (data not shown). Capsaicin treatment stimulated iCGRP release approximately 10-fold above basal levels, and 8-iso PGE₂ augmented this capsaicin-evoked release another 2-fold. Ketorolac pretreatment did not alter the stimulating actions of capsaicin or the sensitizing actions of the isoprostane on iCGRP release. These results suggest that the sensitizing actions of 8-iso PGE₂ on peptide release are not mediated by the production of prostaglandins.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

Our results provide the first evidence that isoprostanes lower nociceptive thresholds to mechanical and thermal stimuli. In addition, we show that these peroxidation products of arachidonic acid augment the excitability and transmitter release from isolated sensory neurons. Together, these results are consistent with the notion that isoprostanes, especially 8-iso PGE₂, produce hyperalgesia, in part, by a direct action on nociceptive sensory neurons. By using isolated sensory neurons, we minimize the possibility that isoprostanes are acting on other cells to produce inflammatory mediators that in turn affect sensory neurons. Furthermore, our findings that ketorolac pretreatment does not attenuate the ability of 8-iso PGE₂ to augment transmitter release supports the notion that some of the actions of isoprostanes are not secondary to production of prostaglandins, agents that have been shown to sensitize sensory neurons (Handwerker, 1976; Schaible and Schmidt, 1988; Nicol and Cui, 1994; Vasko et al., 1994). Finally, it is not likely that the behavioral effects of isoprostanes are secondary to alterations in blood flow to the tissue because both 8-iso PGE₂ and 8-iso PGF₂ have potent actions on the vasculature (Morrow et al., 1990, 1994; Roberts and Morrow, 1997), yet only 8-iso PGE₂ is effective in altering thermal nociception.

Administration of 8-iso PGE₂ produced consistent sensitization of sensory neurons at all endpoints studied; these results are analogous to previous findings regarding the actions of the prostaglandin PGE₂ (Ferreira et al., 1978; Nicol and Cui, 1994; Vasko et al., 1994). In contrast to 8-iso PGE₂, 8-iso PGF₂ administration did not produce consistent results across the various endpoints examined. 8-iso PGF₂ reduced the threshold for mechanical hyperalgesia and increased action potential firing in 6 of 13 isolated sensory neurons, but it did not affect thermal sensitivity or peptide release. Furthermore, the effects of 8-iso PGF₂ were more transient than those of 8-iso PGE₂. One explanation for this difference is that 8-iso PGF₂ only affects a subpopulation of nociceptive sensory neurons, whereas 8-iso PGE₂ has a broader spectrum of action. Indeed, different types of noxious stimuli may activate selective subpopulations of sensory neurons (Lawson, 1994); this could account for the differential responses of the two isoprostanes. It also is possible that within a population of nociceptors, sensitization by 8-iso PGF₂ could vary with different types of noxious stimuli. Indeed, Kumazawa and coworkers proposed that different sensitizing mechanisms exist for noxious thermal versus chemical stimuli because heat-induced sensitivity causes a greater increase in response to subsequent heat compared with bradykinin (Mizumura et al., 1992). Thus, it is feasible that 8-iso PGF₂ could sensitize the same sensory neurons to mechanical but not to thermal nociception.

If isoprostanes are mediators of pain and/or neurogenic inflammation, then it might be expected that that they could be released during tissue trauma and inflammation. To date, measurements of isoprostanes at sites of tissue trauma and inflammation have not been made. Formation of isoprostanes, however, is augmented under a number of conditions of oxidative stress (Reilly et al., 1999). Because tissue injury and inflammation results in an oxidative burst and accumulation of hydrogen peroxide and hydroxyl radicals at the sites of trauma (Kehrer, 1993), one might expect enhanced lipid peroxidation and thus, the formation of isoprostanes. Interestingly, reactive oxygen species have been shown to enhance the firing of sensory neurons (Stahl et al., 1993). Thus, it is intriguing to speculate that a component of their excitatory actions might be mediated by the production of isoprostanes.

It has not been established whether the effects of isoprostanes are mediated through activation of prostaglandin or thromboxane receptors or secondary to activation of unique isoprostane receptors. The effects of 8-iso PGE₂ on canine epithelium are desensitized by pre-exposure to PGE₂ and attenuated by thromboxane receptor antagonists (Elmhurst et al., 1997). 8-iso PGF₂ also partially displaces radioligand binding at thromboxane, prostacycline, PGE₂, and PGF₂ receptors (Kiriyama et al., 1997). These data support the notion that isoprostanes actions involve prostanoid and thromboxane receptors. However, other studies assessing binding and competitive actions of isoprostanes and a thromboxane receptor antagonist suggest that isoprostanes act at unique receptors (Longmire et al., 1994; Yura et al., 1995). Independent of receptor subtypes, however, it is interesting to speculate that the effects of the isoprostanes on sensory neurons are mediated by activation of cAMP and/or the protein kinase C transduction cascades. Indeed, activation of the cAMP transduction cascade or of protein kinase C can sensitize sensory neurons (Cui and Nicol, 1995; Hingtgen et al., 1995; Barber and Vasko, 1996) and can produce hyperalgesia (Ferreira and Nakamura, 1979; Coderre, 1992). In addition, the sensitizing actions of PGE₂ and PGI₂ on sensory neurons are mediated by the cAMP transduction cascade (Cui and Nicol, 1995; Hingtgen et al., 1995). Given the analogous actions of these prostaglandins and 8-iso PGE₂, it seems likely that they could be acting via similar intracellular mechanisms.

Our results have important implications regarding the potential etiologies of hypersensitivity associated with tissue injury and inflammation. Although isoprostanes can be formed as secondary products by cyclooxygenases, this metabolic pathway accounts for only a small portion of the total amount of isoprostanes produced (Pratico et al., 1995; Klein et al., 1997). Because the major component of isoprostane formation is a nonenzymatic pathway through lipid peroxidation, hyperalgesia induced by these agents is not likely to be attenuated by blocking cyclooxygenase activity. Consequently, under conditions where isoprostanes could be produced and released, NSAIDs might have minimal therapeutic effectiveness.

Given our findings that isoprostanes sensitize sensory neurons, additional studies clearly are warranted to assess the potential importance of these compounds in the etiology of pain and inflammation. Furthermore, establishing that isoprostanes modulate excitability of sensory neurons suggests that these eicosanoids should be considered as potential mediators in other instances where oxidative stress alters neuronal function.