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NEUROPHARMACOLOGY
Anesthesiology Research Laboratory 0818 (L.K., J.A.G., T.L.Y); and Department of Pharmacology, University of California, San Diego, La Jolla, California (T.L.Y.)
Received April 5, 2004; accepted June 2, 2004.
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Abstract |
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; O'Brien et al., 2001). The native form of each receptor includes consensus sites for proteolytic cleavage in the extracellular portion of the amino terminus. After cleavage, the remaining amino terminus activates the receptor. A comparable activation is achieved by synthetic peptides with corresponding sequences (Chen et al., 1994). These receptor-derived ligand peptides show a general homology across species (Macfarlane et al., 2001; O'Brien et al., 2001). Once activated, these receptors typically increase intracellular calcium concentrations (Macfarlane et al., 2001). Such increases in calcium initiate many signaling events, including activation of the phospholipase A2-cyclooxygenase cascade (Svensson and Yaksh, 2002). In some cell types, PAR activation has indeed been shown to stimulate prostaglandin production (Neufeld and Majerus, 1983).
Although much work has focused on the peripheral distribution and function of PARs, other work indicates the presence of these receptors in the central nervous system. Reversible binding of radiolabeled human 
-thrombin has been shown in human brain and spinal cord (McKinney et al., 1983). Thrombin binding to cultured murine spinal ventral horn cells has been demonstrated using histological techniques (Means and Anderson, 1986). In situ hybridization and Northern blot analysis revealed substantial expression of PAR 1 mRNA in the rat brain as well as spinal neurons, dorsal root ganglia, and the sciatic nerve (Niclou et al., 1998). An in situ hybridization study found PAR 1 mRNA in the spinal gray matter (Weinstein et al., 1995). PAR 2 has also been found in neurons, with PAR 1 and PAR 2 mRNA found in cultured hippocampal neurons (Smith-Swintosky et al., 1997). PAR 1 and PAR 2 immunoreactivity have both been demonstrated in cells of the dorsal root ganglion (Steinhoff et al., 2000). PAR 3 mRNA was detected by Northern blot analysis of human spinal cord tissue (Ishihara et al., 1997). Recently, reverse transcription-polymerase chain reaction and in situ hybridization demonstrated all four PAR subtypes in cultured hippocampal slices and explants (Striggow et al., 2001). Finally, reverse transcription-polymerase chain reaction and immunohistochemistry demonstrated all four PARs in primary rat astrocyte cultures (Wang et al., 2002a).
The presence of PARs in the central nervous system and the ability of PARs to stimulate prostaglandin synthesis led us to consider whether PAR activation in the spinal cord would also lead to prostaglandin release and nociception. Spinal prostaglandin release is a common feature of many nociceptive stimuli, and inhibition of cyclooxygenase activity blocks their action in animal models of nociception (for review, see Svensson and Yaksh, 2002). Administration of PGE2 produces both hyperalgesia and allodynia in animals (Minami et al., 1994). Accordingly, we hypothesized that activating spinal PARs would produce prostaglandin E2 release. Moreover, those PARs that evoked prostaglandin release would also produce cyclooxygenase-dependent thermal hyperalgesia. Both thrombin and peptides were used to activate PARs. Spinal transmitter release was measured using microdialysis. Nociception was assessed using thermal testing, von Frey filaments, and formalin-induced paw flinching.
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Materials and Methods |
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Preparation. Intrathecal catheters were prepared from polyethylene-10 tubing (0.6 mm outside diameter, 0.3 mm inside diameter; BD Biosciences, Sparks, MD). Tubing was tied in a half-hitch of approximately 3 mm diameter, and the knot was coated with dental acrylic (Dentsply, Milford, DE). After the acrylic dried, the intrathecal side of the tubing was stretched to reduce its diameter. The final product was trimmed to a length of 85 mm.
The intrathecal portion of the dialysis probe consisted of a tubular 4-cm cellulose dialysis fiber (Filtral AN69HF; Cobe Laboratories, Denver, CO) bent in half and connected by its ends to 7 cm of triple lumen polyethylene tubing (Spectranetics, Colorado Springs, CO) using fused silica tubing (Polymicro Technologies, Phoenix, AZ) as stents with methacrylate adhesive (LocTite North America, Rocky Hill, CT). Fine gauge wire (A-M Systems, Carlsborg, WA) inside the dialysis fiber prevented crimping. The center lumen of the polyethylene tubing was used as an injection line. Each of the three lumens was heat fused to short lengths of single lumen polyethylene tubing.
Male Holtzman Sprague-Dawley rats (Harlan, Madison, WI), weighing about 300 to 350 g at the beginning of the experiment, were prepared with lumbar intrathecal injection catheters or dialysis probes. Rats were anesthetized with isoflurane (Abbott Laboratories, North Chicago, IL) in a flow-through chamber. After induction, isoflurane (2–3% in air) was delivered through a nose cone. After shaving and cleaning the scalp, the rat was supported on a pad and secured via ear bars and the nose cone. Sharp dissection was used to separate muscles from the occipital bone and expose the atlantooccipital membrane. A stab blade was used to make an incision in the dura mater. The dialysis probe or intrathecal catheter was then inserted and advanced caudad. Connections were tunneled under the skin to exit over the frontal bones; the free ends were plugged with short lengths of wire (V. Mueller, McGaw Park, IL). Catheters and the injection line of dialysis probes were flushed with saline; the dialysis circuit was flushed with dilute penicillin-streptomycin solution (Invitrogen, Carlsbad, CA). After the incision was closed, rats were placed in a warm chamber to recover from anesthesia. On demonstrating intact neurological function, rats were returned to the vivarium.
Spinal Microdialysis. Dialysis experiments were conducted in conscious rats after 2 or 3 days' recovery from surgery. Previous data suggested that this recovery period resulted in relatively consistent, detectable dialysate concentrations of analytes (Malmberg and Yaksh, 1995). Tubing from the rat was connected to a two-channel fluid swivel (Eicom, Kyoto, Japan; Bioanalytical Systems, West Lafayette, IN; Instech Laboratories, Plymouth Meeting, PA). The effluent was routed to a programmable, refrigerated fraction collector (Eicom). Syringe drivers (Harvard Apparatus Inc., South Natick, MA) delivered artificial cerebrospinal fluid at 10 µl/min. The final composition of the fluid was 151 mM sodium, 2.6 mM potassium, 132 mM chloride, 1.3 mM calcium, 0.9 mM magnesium, 2.5 mM phosphate, and 21 mM carbonate (all salts, EM Scientific, Gibbstown, NJ). The solution was bubbled with 5% CO2 in air until clarity and filtered for sterility. Fractions (10 min) were collected at 4°C and frozen immediately (-20°C for short-term storage, -70°C for long-term storage). Initial experiments and previous studies indicated that analyte levels reach a steady state within 30 min of beginning perfusion (L. Koetzner, M. Marsala, and T. L. Yaksh, unpublished data; Malmberg and Yaksh, 1995). Rats were only tested once to avoid any possibility of carryover effects.
Chemistry: Amino Acid Measurements. Samples were thawed and internal standard (400 pmol of methionine sulfone; Sigma-Aldrich, St. Louis, MO) was immediately added. The samples were dried under vacuum, then reconstituted in methanol/aqueous sodium acetate-triethylamine (all solvents and high-performance liquid chromatography reagents; Fisher Chemical Co., Fair Lawn, NJ) and dried again. Amines were converted to chromogenic derivatives by conjugation with phenylisothiocyanate (5 min at room temperature in methanol/water/triethylamine). The reaction mixture was dried under vacuum and reconstituted in acetonitrile/aqueous sodium phosphate. Samples (30 µl) of this mixture were injected for high-performance liquid chromatography using a 712 autosampler (Waters, Milford, MA). Analytes were separated using a C18 column (5 µm x 25 cm, maintained at 46°C; MetaChem Polaris; MetaChem Technologies, Torrance, CA) and gradient elution with a gradient cleanup step (60 min; 510 pump, 1 ml/min; Waters), controlled by computer. Peaks were detected by monitoring absorbance at 254 nm (0.05 AUFS, 2 samples/s; 1100 detector; Agilent Technologies, Palo Alto, CA). Ratios of analyte peak area to internal standard peak area were calculated for all samples; analyte mass was estimated by comparison to a 400-pmol standard solution. Estimates of aspartate, glutamate, serine, and taurine concentrations were obtained from the same chromatographic separation.
Chemistry: Prostaglandin Analysis. PGE2 concentrations were assayed by competitive enzyme-linked immunosorbent assay using a commercially available kit (Assay Designs 90001; Assay Designs, Ann Arbor, MI). This kit uses a monoclonal antibody to PGE2 (anchored to the plate via anti-IgG antibody) and PGE2-alkaline phosphatase conjugate. The PGE2 antibody has 70% cross-reactivity with PGE1 and 16% cross-reactivity with PGE3; no other cross-reactivities exceeding 2% are known (manufacturer's specifications). After incubation of the reagents and sample, activity bound to the solid phase is measured with paranitrophenylphosphate as absorbance at 450 nm. The amount of analyte in the sample is inferred from displacement of the PGE2-alkaline phosphatase conjugate, and sample values are determined by reference to a standard curve. The linear detection range is generally 40 to 5000 pg/ml sample. Some rats were excluded due to elevated baseline PGE2 concentrations (>1000 fmol/100 µl fraction) or unmeasurably low PGE2 concentrations (<40 fmol/100 µl fraction).
Behavior: Thermal Escape Latency. Hindpaw withdrawal in response to thermal stimulation was measured using a modification of the Hargreaves procedure, as reported previously (Dirig et al., 1997). Briefly, rats were allowed to habituate to a temperature-controlled glass floor. Latency to respond to a heat stimulus, applied to the glass surface under a hind paw, was measured with an electronic timer. Rats were tested 1 wk after catheter implant. Baseline latencies were measured, and stimulus intensity was adjusted to give latencies between 10 and 12 s. Treatments were injected and postinjection latencies were recorded for up to 2 h. Rats were also observed for untoward effects of treatments using a behavior checklist. Repeated dosage of thrombin at 4-d intervals did not produce untoward effects. However, repeated administration of peptides was associated with a broad spectrum of toxicities, with ataxia predominant. Accordingly, peptide treatments were only tested once in each rat.
Behavior: Tactile Allodynia. Tactile allodynia was determined using von Frey filaments presented in a method of limits pattern. The plantar surface of the paw was tested, with a brisk withdrawal of the paw defined as a behavioral response. In addition, an area on the back lateral to the spine at the level of the third lumbar vertebra was tested, with vocalization or rapid orientation of the head to the filament defined as a behavioral response.
Behavior: Formalin-Induced Flinching. Flinching behavior after the intraplantar injection of 50 µl of 0.5% formalin [(v/v) in saline] was counted using the automated device described by Yaksh et al. (2001b). A metal band was affixed to the paw before injection using methacrylate adhesive. After habituation, the rat was placed on an electromagnetic transducer in a polycarbonate cylinder (approx. 15 cm in diameter x 30 cm in height). The signal from the transducer was digitized and signals characteristic of flinching behavior were counted using a proprietary algorithm (Yaksh et al., 2001b).
Treatments. Bovine thrombin (E.C. 3.4.21.5
[EC]
, approx. 75 NIH units/mg; Sigma-Aldrich) was dissolved in saline. Typically, aliquots were frozen. Due to the lability of the enzyme, aliquots were never refrozen or used after more than a 2-wk storage at -20°C. Peptides were selected to mimic the cryptic ligands of rodent PARs (PAR 1, SFLLRN-NH2; PAR 2, SLIGRL; PAR 3, TFRGAP; and PAR 4, GYPGQV; all from Bachem California, Torrance, CA). For some studies, TFLLR (Bachem California) was used as a selective activator of PAR 1 (Blackhart et al., 1996). Peptides and the sodium salt of (+)-ibuprofen (Ethyl Corporation, Orangeburg, SC) were dissolved in saline. COX inhibitors SC 58,560 (selective for COX 1) and SC 58,125 (selective for COX 2) were provided as a gift by Monsanto-Searle (St. Louis, MO). COX inhibitors were dissolved in 10% dimethyl sulfoxide, 66% Cremophor EL (Sigma-Aldrich), 24% saline for systemic delivery. Controls were carried out with corresponding vehicles. For intrathecal injection, treatments were prepared so that the total dose was given in a volume of 10 µl with a 10-µl saline flush. All doses are presented in terms of salt forms.
Data Analysis. Due to the presence of differences in baseline amino acid release, each rat's amino acid microdialysis data are expressed as the percentage of the rat's baseline release. This baseline is the average of the two fractions collected immediately before injection. Differences in baseline PGE2 release were small, so PGE2 data were not normalized. Statistical significance was assessed using analysis of variance on integrated release data (i.e., the sum of postinjection values); post hoc testing used Fisher's protected least significant difference test. Raw data and area over curve data were used for analysis of thermal escape latencies; areas over the curve were calculated using the trapezoidal method. Analysis of variance was generally used with Tukey's multiple comparison test; however, in analysis of raw data for peptide effects, error values were not evenly distributed and group sizes were small. In this subset of studies, Kruskal-Wallis tests were used to eliminate the effects of heteroscedacity. Response thresholds for tactile allodynia testing were calculated as the force necessary to produce a probability of behavioral response of 0.5. Either a statistically significant change in threshold or a decrease of threshold below 4 g was considered allodynia.
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Results |
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Thrombin and Microdialysis. Rats treated with 30 ng of thrombin showed a substantial and long-lasting increase in dialysate PGE2, which peaked between 30 and 90 min after injection (Fig. 1). Analysis of variance revealed a significant treatment effect [F(2,23) = 4.873; p < 0.05], with thrombin-treated rats having higher dialysate PGE2 than vehicle-injected rats (p < 0.05) or uninjected control rats (p < 0.01). Rats in the two control groups did not differ significantly (p = 0.27). Spinal release of amino acids was also measured by microdialysis before and after injection of 100 ng of thrombin (n = 8). None of the amino acids showed a substantial increase after thrombin administration (Fig. 2).
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PAR-Derived Peptides and Microdialysis. The effects of the PAR 1-derived peptide SFLLRN-NH2 were tested at intrathecal doses of 3, 30, and 200 µg. SFLLRN-NH2 produced a substantial, dose-dependent increase in PGE2 release, tending to come in a rapid peak in the first 20 min after injection and a second peak at approximately 1 h after injection (Fig. 3). When the postinjection PGE2 release was summed, the effect of SFLLRN-NH2 was statistically significant [F(3,29) = 8.542; p < 0.001] (Fig. 4). Compared with the vehicle-injected rats, the effects of 30 µg (p < 0.001) and 200 µg(p < 0.01) were significant, whereas those of 3 µg were not (p = 0.72).
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The PAR 2-derived peptide SLIGRL was tested at intrathecal doses of 3, 30, and 200 µg. SLIGRL increased PGE2 release with a biphasic time course (Fig. 5). Integrated postinjection PGE2 release showed a biphasic and statistically significant effect of SLIGRL [F(3,26) = 33.172; p < 0.001] (Fig. 4). Both 3 and 30 µg of SLIGRL stimulated more release than vehicle (p < 0.01 and p < 0.001, respectively), but the effect of 200 µg was not statistically significant (p = 0.25).
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The PAR 3-derived peptide TFRGAP and the PAR 4-derived GYPGQV were each tested at 200 µg (Fig. 6). Compared with vehicle, no statistically significant treatment effects were observed [F(2,20) = 0.521; p = 0.60].
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Thrombin and Behavior. Thrombin-induced thermal hyperalgesia was tested using withdrawal latencies in a modified Hargreaves device. Pilot studies indicated that intrathecal doses of up to 10 ng of thrombin were without effect. At doses from 300 ng to 1 µg, rats developed lethargy combined with chromodacyorrhea, ptosis, and piloerection. These high-dose effects are reminiscent of the behavior seen with high doses of PGE2 (Minami et al., 1994; C. I. Svensson and T. L. Yaksh, unpublished data). For full studies, thermal escape latencies were tested using thrombin doses from 30 to 300 ng and compared with 300 ng of bovine serum albumin (n = 7 observations/treatment). Treatment with 30 ng of thrombin decreased paw withdrawal latencies relative to baseline and relative to the control treatment (Fig. 7A). The magnitude of the hyperalgesic effect peaked 60 min after injection and remained substantial at 90 min; at 2 h after injection, latencies were not different from those of control rats (data not shown). This effect was dose-dependent [Fig. 7B; F(3,24) = 3.304; p < 0.05], because rats treated with 300 ng of thrombin showed a general suppression of behavior, including an increase in paw withdrawal latency.
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Tactile sensitivity was measured using von Frey filaments before and 20, 40, or 60 min after the intrathecal injection of 100 ng of thrombin (n = 6). The plantar surface of the paw and a lumbar dorsal paramedian site were both tested. Allodynia did not develop at either site; paw withdrawal thresholds never decreased below 11.6 g, and dorsum response thresholds never decreased below 14.2 g after injection.
The spontaneous flinching response to intraplantar formalin injection was tested in rats receiving either 100 ng of thrombin or vehicle (n = 7/group). Pretreatments were injected via intrathecal catheters 15 min before formalin injection [0.5% (v/v)]. Neither phase 1 flinching behavior (the first 9 min after formalin injection) nor phase 2 flinching behavior (from 10 to 60 min after formalin injection) was affected (data not shown).
PAR-Derived Peptides and Behavior. Intrathecal injection of saline or 30 µg of SFLLRN-NH2 or SLIGRL (n = 6/treatment) resulted in reductions in hindpaw withdrawal latency (i.e., hyperalgesia) at the limit of statistical significance (Kruskal-Wallis H = 5.66, df = 2, p = 0.059). Intrathecal injection of the PAR 1-selective peptide TFLLR did not produce thermal hyperalgesia (data not shown). In rats pretreated with (+)-ibuprofen (30 µg intrathecal, 20 min before peptide injection), no significant effect of SFLLRN-NH2 or SLIGRL administration was observed (Fig. 8B; H = 1.21, df = 2, p = 0.55). The effects of SLIGRL were investigated further with isozyme-specific inhibitors. Both the COX 1 inhibitor (SC 58,560) and the COX 2 inhibitor (SC 58,125) blocked the thermal hyperalgesia produced by SLIGRL (Fig. 8C). Areas over the curve were significantly different [F(2,15) = 8.09, p < 0.005], with significant effects of both SC 58,560 (Tukey's q = 4.97 versus saline, p < 0.01) and SC 51,245 (q = 4.88 versus saline, p < 0.01).
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; O'Brien et al., 2001; Svensson and Yaksh, 2002) and evokes prostaglandin production (Neufeld and Majerus, 1983). The CNS and spinal cord express all of the proteins necessary for PAR signaling, including all currently identified PARs (Ishihara et al., 1997; Niclou et al., 1998; Steinhoff et al., 2000; Wang et al., 2002a), proteases (Dihanich et al., 1991; Chen et al., 1995; Scarisbrick et al., 1997, 2001) and regulators of protease activity (Niclou et al., 1998). Accordingly, we sought to determine which spinal PARs might be coupled to the formation of PGE2. Activating Spinal PARs Evokes PGE2 Release. Using the intrathecal loop dialysis system, we examined spinal PAR effects on extracellular concentrations of PGE2. Intrathecal thrombin resulted in PGE2 release with no effect on aspartate, glutamate, serine, or taurine (Figs. 1 and 2). PGE2 release was observed after intrathecal delivery of agonist peptides derived from PAR 1 and PAR 2 (Figs. 3, 4, and 6). In contrast, peptides derived from PARs 3 and 4 had no effect, even at high doses (Figs. 5 and 6). The initial time course of prostaglandin release suggests that de novo cyclooxygenase synthesis is not required, i.e., that spinal cyclooxygenases supporting PAR signaling are constitutively expressed. The failure of PAR 3 and PAR 4 peptides to stimulate PGE2 production demonstrates the specificity of the effects of the PAR 1 and PAR 2 peptides.
The data from these studies strongly suggest that, at the spinal level, PAR 2 is involved. The data are less clear regarding the role of PAR 1. Ligand specificity is currently a limitation in the interpretation of PAR studies in general. Few PAR ligands have been validated across the entire family of PARs (Macfarlane et al., 2001; O'Brien et al., 2001). Both SLIGRL and SFLLRN will activate a homogenous population of PAR 2, although PAR 1 is only activated by SFLLRN (Lerner et al., 1996)). Thrombin shows a clear preference for PAR 1 (Nystedt et al., 1994; Blackhart et al., 1996). Whereas initial reports suggested PAR 3 was inactive, several studies have reported efficacy of TFRGAP at PAR 3 (Wang et al., 2002a,b). The current biochemical literature strongly suggests that PAR 3- and PAR 4-derived peptides have severalfold lower affinity for their receptors than do PAR 1- and PAR 2-derived peptides do for their receptors (Xu et al., 1998). Thus, negative findings in these experiments with TFRGAP and GYPGQV strongly suggest that PAR 3 and PAR 4 are not involved.
Activating Spinal PARs Evokes Hyperalgesia. Consistent with the increase in PGE2 release, intrathecal thrombin and PAR 2-derived peptides caused thermal hyperalgesia. This hyperalgesia was blocked by ibuprofen and both COX 1- and COX 2-selective inhibitors. Although thrombin and the PAR 1-derived peptide SFLLRN-NH2 produced thermal hyperalgesia, the more PAR 1-selective peptide TFLLR did not. Consistent with these behavioral observations, the dialysis data suggest that PAR 2 and, with lesser certainty, PAR 1 are involved. More specific ligands will be necessary to conclusively define the role of spinal PAR 1.
Previous work has emphasized that the effect of PGE2 may reflect some combination of enhanced release of transmitters from primary afferents, depolarization of second order afferents, or reduced activation of inhibitory interneurons (for review, see Svensson and Yaksh, 2002). The locus of PAR activity, however, is not clear. Thrombin resulted in a significant increase in PGE2 but not amino acids, implicating mechanisms other than activation of primary afferents. This selectivity is unusual in our experience with nociceptive spinal activators such as intraplantar formalin (Malmberg and Yaksh, 1995) and intrathecal dynorphin (Koetzner et al., 2004). A lack of primary afferent activity contrasts with the activity of peripheral nerve PARs. For example, in superfused rat dorsal spinal cord tissue, trypsin, tryptase, and SLIGRL stimulate release of substance P (Steinhoff et al., 2000). Intrathecal administration of neurokinin receptor 1 antagonists blocks hyperalgesia after intraplantar or intracolonic administration of SLIGRL (Vergnolle et al., 2001; Coelho et al., 2002).
Role of COX 1 and COX 2. The present observations show that both COX 1 and COX 2 inhibitors will reduce PAR-evoked hyperalgesia and establish a crucial downstream linkage with the cPLA2-COX cascade. In many systems, COX-mediated hyperalgesia seems to be produced exclusively by COX 2, e.g., after intraplantar carrageenan and intrathecal substance P (Yaksh et al., 2001a). The limited role of COX 1 in spinal PGE2 release has been surprising. The absence of an effect has been suggested to reflect different distributions or differences in substrate requirements for the two isozymes. More recently, there has been a growing appreciation of the role for COX 1, as after nerve injury (Ma et al., 2002) or after spinal delivery of dynorphin (Koetzner et al., 2004). Spinal PAR 2 activation may also involve the downstream events that are a critical for these other COX 1-activating stimuli.
Physiological Significance of PAR Activation. The physiological significance of PAR activation in the periphery can be inferred from experiments that have blocked the protease-PAR interaction. For example, soybean trypsin inhibitor pretreatment nearly eliminated carrageenan-induced hyperalgesia and inflammation (Van Arman et al., 1966). Similarly, mice with a targeted disruption of the PAR 2 gene show a diminished response to intraplantar formalin or the mast cell degranulator compound 48/80 (Vergnolle et al., 2001). For PARs in the brain and spinal cord, there are two possible sources of proteases. Blood-derived enzymes (e.g., thrombin) are likely to play a role in cases of blood-brain barrier disruption; this could involve penetrating injury, but diminished blood-brain barrier function may also be present in conditions ranging from stress to status epilepticus (Gingrich and Traynelis, 2000). Small but pervasive breaches of the blood-brain barrier may also be a feature of dural puncture headache (Jankowski, 2002). However, a more intriguing possibility involves the production of serine proteases by CNS tissue.
Several laboratories have demonstrated a variety of serine proteases in central nervous system tissues and suggested their physiological relevance. Prothrombin mRNA has been demonstrated in rat brain; additionally, prothrombin mRNA has been found in dorsal root ganglia (Dihanich et al., 1991). Although no reports to date have identified trypsin in the brain or spinal cord, a number of trypsin homologs have been identified. Neuropsin mRNA is found in the hippocampus and cortex, and mRNA increased substantially after electrical kindling (Chen et al., 1995). MSP is a trypsin homolog cloned from rat spinal cord (Scarisbrick et al., 1997) with broad substrate specificity; the probable cleavage sequence fits the cryptic PAR 2 peptide (Blaber et al., 2002). Kainate administration increased MSP expression in all areas of the spinal cord, suggesting a link between excitatory amino acid receptors and MSP (Scarisbrick et al., 1997). A more recent study has found MSP mRNA and immunoreactivity in human dorsal root ganglia and spinal cord (Scarisbrick et al., 2001). Finally, a serine protease with trypsin-like substrate preference and inhibitor sensitivity has been cloned from rat brain; it activates PAR 2 on glioblastoma cells (Sawada et al., 2000). These data, combined with reports of protease regulators in the central nervous system (Niclou et al., 1998), suggest that the CNS is capable of regulated proteolytic signaling through PARs.
PARs are a new class of receptors with a relatively undeveloped pharmacology. PARs have a complex regulatory cycle; for example, PAR 2 calcium signaling is polyphasic, with an initial peak and a later plateau (Böhm et al., 1996). PAR activity couples to cyclooxygenases, which are subject to their own complex regulation (Wu et al., 1999). The vagaries of prostanoid effects on behavior are well known (Svensson and Yaksh, 2002). These uncertainties force us to make certain speculations in the interpretation of our data. We attribute the increased response latency after high doses of thrombin (Fig. 7) to a general suppression of behavior rather than analgesia on the basis of diminished locomotor behavior and markers of stress observed after high-dose thrombin (see Results; data not shown) that are typical of high-dose PGE2 effects (Svensson and Yaksh, 2002). The complexities of the PGE2 response (i.e., biphasic dose-response and time course; Figs. 3, 4, 5, 6) could be a reflection of the regulation of PARs and cyclooxygenases in the spinal cord. However, the state of our understanding of both PAR and cyclooxygenase function means that this attribution is purely speculative.
In summary, we have demonstrated that stimulation of spinal PARs 1 and 2 produces cyclooxygenase-dependent thermal hyperalgesia. Further work will be necessary to map out the signal transduction pathways that link PARs with cyclooxygenase activity. PAR-stimulated calcium flux may drive kinase activity; indeed, it has long been known that cellular tyrosine phosphorylation increases after thrombin treatment (Ferrell and Martin, 1988). This suggests a role for mitogen-activated protein kinases (Wang et al., 2002b), a question that will be resolved in future experiments.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: PAR, protease-activated receptor; COX, cyclooxygenase; SC 58,125, 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole; SC 58,560, 5-(4-fluorophenyl)-1-[4-(methylsulfonyl)phenyl]-3-(trifluoromethyl)-1H-pyrazole; CNS, central nervous system; PGE, prostaglandin; MSP, myelencephalon-specific protease.
1 Current address: Department of Neuropharmacology, Purdue Pharma L.P., Cranbury, NJ 08512. 
Address correspondence to: Dr. Tony L. Yaksh, Anesthesiology Research Laboratory–0818, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0818. E-mail: tyaksh{at}ucsd.edu
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References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Blaber SI, Scarisbrick IA, Bernett MJ, Dhanarajan P, Seavy MA, Jin Y, Schwartz MA, Rodriguez M, and Blaber M (2002) Enzymatic properties of rat myelencephalon-specific protease. Biochemistry 41: 1165-1173.[CrossRef][Medline]
Blackhart BD, Emilsson K, Nguyen D, Teng W, Martelli AJ, Nystedt S, Sundelin J, and Scarborough RM (1996) Ligand cross-reactivity within the protease-activated receptor family. J Biol Chem 271: 16466-16471.
Böhm SK, Khitin LM, Grady EF, Aponte G, Payan DG, and Bunnett NW (1996) Mechanisms of desensitization and resensitization of proteinase-activated receptor-2. J Biol Chem 271: 22003-22016.
Chen J, Ishii M, Wang L, Ishii K, and Coughlin SR (1994) Thrombin receptor activation. Confirmation of the intramolecular tethered liganding hypothesis and discovery of an alternative intermolecular liganding mode. J Biol Chem 269: 16041-16045.
Chen Z-L, Yoshida S, Kato K, Momota Y, Suzuki J, Tanaka T, Ito J, Nishino H, Aimoto S, Kiyama H, et al. (1995) Expression and activity-dependent changes of a novel limbic-serine protease gene in the hippocampus. J Neurosci 15: 5088-5097.[Abstract]
Coelho A-M, Vergnolle N, Guiard B, Fioramonti J, and Bueno L (2002) Proteinases and proteinase-activated receptor 2: a possible role to promote visceral hyperalgesia in rats. Gastroenterology 122: 1035-1047.[CrossRef][Medline]
Dihanich M, Kaser M, Reinhard E, Cunningham D, and Monard D (1991) Prothrombin mRNA is expressed by cells of the nervous system. Neuron 6: 575-581.[CrossRef][Medline]
Dirig DM, Salami A, Rathbun ML, Ozaki GT, and Yaksh TL (1997) Characterization of variables defining hindpaw withdrawal latency evoked by radiant thermal stimuli. J Neurosci Methods 76: 183-191.[CrossRef][Medline]
Ferrell JE Jr and Martin GS (1988) Platelet tyrosine-specific protein phosphorylation is regulated by thrombin. Mol Cell Biol 8: 3603-3610.
Gingrich MB and Traynelis SF (2000) Serine proteases and brain damage - is there a link? Trends Neurosci 23: 399-407.[CrossRef][Medline]
Ishihara H, Connolly AJ, Zeng D, Kahn ML, Zheng Y-W, Timmons C, Tram T, and Coughlin SR (1997) Protease-activated receptor 3 is a second thrombin receptor in humans. Nature (Lond) 386: 502-506.[CrossRef][Medline]
Jankowski CJ (2002) Neuraxial anesthetic techniques, in Textbook of Regional Anesthesia (Raj PP ed) pp 829-852, Churchill Livingstone, New York.
Koetzner L, Hua X-Y, Lai J, Porreca F, and Yaksh TL (2004) Nonopioid actions of intrathecal dynorphin evoke spinal excitatory amino acid and prostaglandin E2 release mediated by cyclooxygenase-1 and -2. J Neurosci 24: 1451-1458.
Lerner DJ, Chen M, Tram T, and Coughlin SR (1996) Agonist recognition by proteinase-activated receptor 2 and thrombin receptor. Importance of extracellular loop interactions for receptor function. J Biol Chem 271: 13943-13947.
Ma W, Du W, and Eisenach JC (2002) Role for both spinal cord COX-1 and COX-2 in maintenance of mechanical hypersensitivity following peripheral nerve injury. Brain Res 937: 94-99.[CrossRef][Medline]
Macfarlane SR, Seatter MJ, Kanke T, Hunter GD, and Plevin R (2001) Proteinase-activated receptors. Pharmacol Rev 53: 245-282.
Malmberg AB and Yaksh TL (1995) Cyclooxygenase inhibition and the spinal release of prostaglandin E2 and amino acids evoked by paw formalin injection: a microdialysis study in unanesthetized rats. J Neurosci 15: 2768-2776.[Abstract]
McKinney M, Snider RM, and Richelson E (1983) Thrombin binding to human brain and spinal cord. Mayo Clin Proc 58: 829-831.[Medline]
Means ED and Anderson DK (1986) Thrombin interactions with central nervous system tissue and implications of these interactions. Ann NY Acad Sci 485: 314-322.[CrossRef][Medline]
Minami T, Uda R, Horiguchi S, Ito S, Hyodo M, and Hayaishi O (1994) Allodynia evoked by intrathecal administration of prostaglandin E2 to conscious mice. Pain 57: 217-223.[CrossRef][Medline]
Neufeld EJ and Majerus PW (1983) Arachidonate release and phosphatidic acid turnover in stimulated human platelets. J Biol Chem 258: 2461-2467.
Niclou SP, Suidan HS, Pavlik A, Vejsada R, and Monard D (1998) Changes in the expression of protease-activated receptor 1 and protease nexin-1 mRNA during rat nervous system development and after nerve lesion. Eur J Neurosci 10: 1590-1607.[CrossRef][Medline]
Nystedt S, Emilsson K, Wahlestedt C, and Sundelin J (1994) Molecular cloning of a potential proteinase activated receptor. Proc Natl Acad Sci USA 91: 9208-9212.
O'Brien PJ, Molino M, Kahn M, and Brass LF (2001) Protease activated receptors: theme and variations. Oncogene 20: 1570-1581.[CrossRef][Medline]
Sawada K, Nishibori M, Nakaya N, Wang Z, and Saeki K (2000) Purification and characterization of a trypsin-like serine proteinase from rat brain slices that degrades laminin and type IV collagen and stimulates protease-activated receptor-2. J Neurochem 74: 1731-1738.[CrossRef][Medline]
Scarisbrick IA, Isackson PJ, Ciric B, Windebank AJ, and Rodriguez M (2001) MSP, a trypsin-like serine protease, is abundantly expressed in the human nervous system. J Comp Neurol 431: 347-361.[CrossRef][Medline]
Scarisbrick IA, Towner MD, and Isackson PJ (1997) Nervous system-specific expression of a novel serine protease: regulation in the adult rat spinal cord by excitotoxic injury. J Neurosci 17: 8156-8168.
Smith-Swintosky VL, Cheo-Isaacs CT, D'Andrea MR, Santulli RJ, Darrow AL, and Andrade-Gordon P (1997) Protease-activated receptor-2 (PAR-2) is present in the rat hippocampus and is associated with neurodegeneration. J Neurochem 69: 1890-1896.[Medline]
Steinhoff M, Vergnolle N, Young SH, Tognetto M, Amadesi S, Ennes HS, Trevisani M, Hollenberg MD, Wallace JL, Caughey GH, et al. (2000) Agonists of proteinase-activated receptor 2 induce inflammation by a neurogenic mechanism. Nat Med 6: 151-158.[CrossRef][Medline]
Striggow F, Riek-Burchardt M, Kiesel A, Schmidt W, Henrich-Noack P, Breder J, Krug M, Reymann KG, and Reiser G (2001) Four different types of protease-activated receptors are widely expressed in the brain and are up-regulated in hippocampus by severe ischemia. Eur J Neurosci 14: 595-608.[CrossRef][Medline]
Svensson CI and Yaksh TL (2002) The spinal phospholipase-cyclooxygenase-prostanoid cascade in nociceptive processing. Annu Rev Pharmacol Toxicol 42: 553-583.[CrossRef][Medline]
Van Arman CG, Nuss GW, Winter CA, and Flataker L (1966) Proteolytic enzymes as mediators of pain, in Pharmacology of Pain (Eg P ed) pp 25-32, Pergamon Press, Oxford.
Vergnolle N, Bunnett NW, Sharkey KA, Brussee V, Compton SJ, Grady EF, Cirino G, Gerard N, Basbaum AI, Andrade-Gordon P, et al. (2001) Proteinase-activated receptor-2 and hyperalgesia: a novel pain pathway. Nat Med 7: 821-826.[CrossRef][Medline]
Wang H, Ubl JJ, and Reiser G (2002a) Four subtypes of protease-activated receptors, co-expressed in rat astrocytes, evoke different physiological signaling. Glia 37: 53-63.[CrossRef][Medline]
Wang H, Ubl JJ, Stricker R, and Reiser G (2002b) Thrombin (PAR-1)-induced proliferation in astrocytes via MAPK involves multiple signaling pathways. Am J Physiol 283: C1351-C1364.
Weinstein JR, Gold SJ, Cunningham DD, and Gall CM (1995) Cellular localization of thrombin receptor mRNA in rat brain: expression by mesencephalic dopaminergic neurons and codistribution with prothrombin mRNA. J Neurosci 15: 2906-2919.[Abstract]
Wu G, Wei C, Kulmacz RJ, Osawa Y, and Tsai A-L (1999) A mechanistic study of self-inactivation of the peroxidase activity in prostaglandin H synthase-1. J Biol Chem 274: 9231-9237.
Xu W-F, Andersen H, Whitmore TE, Presnell SR, Yee DP, Ching A, Gilbert T, Davie EW, and Foster DC (1998) Cloning and characterization of human protease-activated receptor 4. Proc Natl Acad Sci USA 95: 6642-6646.
Yaksh TL, Dirig DM, Conway CM, Svensson C, Luo ZD, and Isakson PC (2001a) The acute antihyperalgesic action of nonsteroidal, anti-inflammatory drugs and release of spinal prostaglandin E2 is mediated by the inhibition of constitutive spinal cyclooxygenase-2 (COX-2) but not COX-1. J Neurosci 21: 5847-5853.
Yaksh TL, Ozaki G, McCumber D, Rathbun M, Svensson C, Malkmus S, and Yaksh MC (2001b) An automated flinch detecting system for use in the formalin nociceptive bioassay. J Appl Physiol 90: 2386-2402.
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