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NEUROPHARMACOLOGY
Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas
Received May 13, 2005; accepted June 22, 2005.
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Abstract |
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S35 functional binding assays stimulated by an NK-1 selective agonist. Results showed that formalin evoked a transient, bilateral decrease in the maximal functional response to 35% of control in the treated side at 24 h postinjection and as much as a 10-fold leftward shift in the EC50 of the agonist at 12 to 96 h postinjection. These results provide novel evidence that peripheral nociceptive activation promotes a central mechanism of hyperalgesia through increased functional sensitivity of NK-1 receptors in the spinal cord dorsal horn.
; Jacques et al., 1989; Tsuchida et al., 1990; Hershey et al., 1991; Ansel et al., 1996; McCarson, 1999) and is concentrated in the superficial lamina of the spinal cord dorsal horn (Tsuchida et al., 1990; Hershey et al., 1991). Our previous results demonstrated elevated levels of mRNA for both the NK-1 receptor and its endogenous peptide ligand substance P (SP) during peripheral inflammation induced by formalin or complete Freund's adjuvant (CFA) (McCarson and Krause, 1994, 1995, 1996; McCarson, 1999). Although an activity-dependent increase in NK-1 mRNA expression may contribute to sensory sensitization (McCarson and Krause, 1994), the net contribution of increased NK-1 mRNA levels to receptor protein production and, more importantly, receptor functional coupling, has not yet been established.
The NK-1 receptor and SP have been widely implicated in nociceptive mechanisms (Radhakrishnan and Henry, 1991; Sakurada et al., 1993; De Felipe et al., 1998). During activation, SP binds to NK-1 and activates G-protein complexes. Upon activation, the G
q/11 subunit undergoes an obligatory exchange of GDP for GTP, dissociates from the 
/ duplex, and subsequently activates phospholipase C (Krause et al., 1992). Hydrolysis of the GTP allows reassociation with the / duplex and formation of a fresh G-protein complex (Collins et al., 1992). Sufficiently intense activation of NK-1 receptors results in clustering of NK-1 receptors on the cell surface (Grady et al., 1995; Mantyh et al., 1995) and subsequent receptor-mediated endocytosis. It has been debated whether the resulting intracellular vesicles contain bound agonist ligand; the internalization of NK-1 receptors occurs within the first 2 min after injection, and receptors are not fully recycled or replaced in the membrane until 90 min postinjection (Wang and Marvizon, 2002). Importantly, the coupling state of the internalized receptors is unclear. Current interpretations suggest that internalization and deactivation of available receptors result in desensitization to further NK-1 agonist application (Ferguson, 2001). However, if the internalized vesicle contains both SP and coupled receptors, superactivation of the second messenger cascade could occur and contribute mechanistically to overactivity and/or sensitization of the cell. Thus, the functional capacity of NK-1 receptors is likely to be markedly different during persistent activation and may be altered for very long periods after peripheral injury. These alterations of the function of the NK-1 receptor are measurable by its coupling state and may contribute greatly to the plasticity of nociceptive pathways.
This study quantified dynamic changes in NK-1 receptor gene expression for 4 days after peripheral inflammation using solution-hybridization nuclease protection assays combined with concurrent measurement of the function of NK-1 receptors using a ligand-stimulated GTPS35 binding assay that measures function of the receptor by incorporating a radiolabeled nonhydrolyzable GTPS35 onto activated G subunits at the point of dissociation from the / duplex. After activation, the G-protein becomes locked and measurable by autoradiography and densitometry (Sovago et al., 2001). The selective NK-1 receptor agonist Sar9Met11(O)2 substance P (smSP; Lew et al., 1990) was used to activate NK-1 receptors in the ex vivo tissue sections. This technique quantifies the number of functional receptors by analyzing Emax values and receptor G-protein coupling affinity through analysis of EC50. The results of this study directly quantify pain-evoked dynamic alterations in NK-1 receptor coupling in the context of ongoing up-regulation of spinal NK-1 receptor gene expression during inflammation-evoked behavioral hyperalgesia.
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). All procedures were performed as outlined in the Association for Assessment and Accreditation of Laboratory Animal Care Guide for Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee at the University of Kansas Medical Center.
Behavioral Measurements. Thermal withdrawal latencies were measured with a Thermal Analgesimeter (University of California, San Diego, CA). Freely moving rats were placed in a Plexiglas chamber, and a high-intensity light beam was focused on the plantar surface of a hind paw or tail as a noxious thermal stimulus (Dirig et al., 1997). Baseline measurements of both hind paws and tail were taken for each animal for 2 days before injection and at 1, 2, 4, 6, 24, 48, 72, and 96 h after injection to measure thermal hyperalgesia or hypoalgesia. Von Frey monofilaments (Stoelting, Inc., Wood Dale, IL) of graded bending forces (2.6–522 mN) were applied to plantar and dorsal aspects of the hind paws of unrestrained rats placed in a Plexiglas chamber with a wire mesh grid bottom to quantify mechanical thresholds. Monofilaments were applied perpendicular to the hind paw surface with sufficient force to cause a slight bending of the filament in increasing order of intensity until the rat responded by vocalization or withdrawal of the paw. Mechanical stimulation was repeated three times at 5- to 10-min intervals, with randomization of order of testing for each paw (adapted from Brennan et al., 1996). Monofilament thresholds were converted to grams of force using the manufacturer's table. At time of sacrifice, hind paws were removed just above the tibio-tarsal joint and weighed to measure edema. Behavioral measurements were conducted by an experimenter blind to animal treatments; the results are presented as mean ± S.E.M. for all animals within a treatment group. Significance was determined using analysis of variance software (SuperANOVA; Abacus Concepts, Berkeley, CA, or SYSTAT; Systat Software, Inc., Point Richmond, CA) with Fisher's PLSD, Dunnett's, or Wilcoxon post hoc tests.
NK-1 Receptor-Stimulated GTPS35 Binding Assays. NK-1 receptor-stimulated GTPS35 binding assays in rat spinal cord membrane preparations were developed based on assays previously established for the 5-hydroxytryptamine1A (Alper and Nelson, 1998) and other G-protein-coupled receptors (Lazareno, 1997). The lumbar enlargement of the spinal cord was dissected and encased in a gelatin capsule containing TBS Freezing Medium (Triangle Biomedical Sciences, Durham, NC). The capsule was snap-frozen in methyl butane. Spinal cord blocks were subsequently stored at -80°C for less than 96 h, at which time they were sectioned on a Leica Cryostat. Fifty-micron-thick sections were cut and mounted on gelatin-coated glass slides. Each slide contained six individual sections from one animal. Slides were stored at -80°C for less than 1 week until the GTPS35 binding assay was performed (Alper and Nelson, 1998). Three slides (18 individual sections) were assayed at each assay condition for each animal. Samples from at least one animal from every treatment group were assayed simultaneously. Sections were equilibrated in assay buffer (30 mM MgCl2, 150 mM NaCl, 2.7 mM KCl, and 37.5 mM HEPES, pH 7.4) for 10 min and then in 2 mM GDP for 15 min both at room temperature. Agonist-stimulated binding was then performed with 2 mM GDP and 0.1 nM GTPS35 (GE Healthcare, Little Chalfont, Buckinghamshire, UK) and one of four serial dilutions ranging from 45 nM to 45 µM smSP (Sigma-Aldrich) in water treated with a peptidase inhibitor cocktail containing 0.170 mg/ml bacitracin, 17 µg/ml leupeptin, 17 µg/ml chymostatin, and 0.850 mg/ml bovine serum albumin. Drug was omitted and replaced with either peptidase-treated water for basal determinations or 10 µM unlabeled GTPS in water for nonspecific binding determinations. Antagonist competition assays were performed with 4.5 µM smSP in the presence of the NK-1 receptor antagonist L-733,060 (Tocris Cookson Inc., Ellisville, MO) in concentrations ranging from 0.01 to 10 µM (Seabrook et al., 1996). Slides were incubated in binding conditions at 37°C for 45 min and then rinsed twice with ice-cold 50 mM Tris-Cl, pH 7.4, for 2 min and twice with room temperature deionized water for 2 min. Slides were air-dried and exposed to Kodak X-OMAT autoradiographic film (Sigma-Aldrich) for 48 to 72 h. Digital images of the resulting autoradiograms were captured using a computer controlled digital camera and analyzed using Scion Image (Scion Corporation, Frederick, MD). Densitometry was performed by measuring the mean number of pixels in lamina I of each left and right dorsal horn and subtracting the mean dorsal column background value. Percent stimulation over basal was calculated using the following equation: [[(((DH dose value) - (column dose value)) - nonspecific binding)/(((DH basal value) - (column basal value)) - nonspecific binding)] x100] - 100. Concentration-response curves were generated, and values for maximal stimulation (Emax) and half-maximal stimulating concentration (EC50) were determined. Tabulated binding parameters represent arithmetic means of individual Emax and EC50 values calculated from separate binding curves for each subject (Sigma Plot; RockWare Inc., Golden, CO). However, the graphical representation shows a single sigmoidal curve generated by Sigma Plot for all subjects simultaneously; hence, the graphical representation underestimates individually determined parameters. The right side of naive rats was used as control unless stated otherwise; significance was determined using ANOVA with Fisher's PLSD.
Assays of NK-1 Receptor Gene Expression. Lumbar spinal cord samples were dissected into dorsal quarters, and left and right total RNA samples were isolated and assayed separately for NK-1 receptor and -actin mRNAs using solution hybridization-nuclease protection assays as described previously (Krause et al., 1989; McCarson, 1999). Samples of 15 to 50 µg of total RNAs were assayed for NK-1 receptor, or 5 µg of total RNA was assayed for -actin mRNA levels. Aliquots of 20 to 100 pg of cRNA quantitation standards were used to generate a standard curve, and Escherichia coli tRNA was used as a negative control. Densitometric images of the resulting denaturing gels were generated using a GE Healthcare PhosphorImager SF and analyzed using IP Lab Gel (Signal Analytics Corporation, Vienna, VA). Data values are reported as picograms of NK-1R mRNA/nanograms of -actin mRNA (mean ± S.E.M.). Data were analyzed using ANOVA with Fisher's tests used for post hoc comparisons, with significance considered to be p 
0.05. Correlation analyses across time points and biochemical and behavioral endpoints were conducted using parametric models to assess correlation coefficients (r2) between measures for mRNA levels, behaviors, and receptor function. The degree of interaction between measures was determined by two-factor ANOVA, with interactions of p 0.05 considered significant. Pearson correlation coefficients were determined and linear regression analyses were performed using Sigma Plot.
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Expression of the NK-1 receptor gene was quantified at several late time points following peripheral inflammatory stimuli. Figure 3 shows that, in the ipsilateral dorsal horn of the spinal cord, unilateral peripheral formalin injection resulted in a significant (approximately 2-fold) increase in NK-1 receptor mRNA levels that was apparent at 2 h and lasted at least 96 h. Expression levels of the NK-1 receptor gene were also slightly, although not significantly, increased in the contralateral spinal cord dorsal horn.
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Initial characterization of NK-1 receptor function after inflammation began with control, formalin (24-h), and CFA (4-day) groups only. Representative autoradiograms showing nonspecific, basal, and stimulated assay conditions are shown in Fig. 4, revealing a robust stimulation of GTPS35 binding in the superficial laminae of the lumbar dorsal horn. Agonist stimulation of NK-1 receptors with smSP revealed a concentration-dependent increase in G-protein activation in the dorsal horn of the rat spinal cord (Fig. 5A). Formalin-induced inflammatory nociception evoked a bilateral decrease in the Emax to approximately one-half of the control maximum and a simultaneous unilateral leftward shift in the EC50 after 24 h (Fig. 5B). The significant shift in the EC50 was on the order of 10-fold on the treated side. On the contralateral side, a slight shift was seen but was not significant at this time point. The concentration-response curve for CFA-treated animals was similar to the curve for control animals (data not shown). Inflammation induced by CFA evoked shifts in the EC50 that were bilateral and not significant at this time point. However, the decrease in the Emax on the treated side was significantly different from control (Fig. 5B).
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To test the specificity of the agonist, three naive control animals were used to generate a competition curve of 4.5 µM smSP in the presence of the potent selective nonpeptide NK-1 receptor antagonist L-733,060 (Seabrook et al., 1996). As seen in Fig. 6, the highest concentration of antagonist used (10 µM) was able to completely block the stimulation by 4.5 µM smSP. Stimulation of NK-1 receptors with 4.5 µM smSP evoked a maximal response of 233.22 ± 54.53% stimulation over basal, and the addition of 10 µM L-733,060 decreased the maximal response to 4.15 ± 3.69% stimulation over basal. The NK-1 antagonist produced an identical blockade of smSP-evoked stimulation in ex vivo spinal cord sections of animals tested either 24 h after formalin injection or 4 days following CFA.
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Once changes in NK-1 receptor function were characterized at these time points, a time course after formalin injection was performed to determine the series of events and duration of changes initially seen at 24 h. In this phase of the study, four control animals were used and three to five animals each were used for time points of 1, 6, 12, 24, and 96 h postformalin. At 1 h, the Emax of both contralateral and ipsilateral sides was significantly reduced (Fig. 7). Values for Emax rebounded toward baseline before declining again at 12 to 24 h. Measurement at 96 h shows that Emax values again return toward preinjection levels, although treated-side values remain significantly lower than control. Similar but less intense changes in the number of functionally coupled NK-1 receptors are evident on the contralateral side as well. Treatment with formalin decreased the EC50 of smSP on the treated side but had no significant effect on the untreated side (Fig. 8). This decrease was significant at 12 h and was maintained throughout the duration of the time course out to 96 h postformalin.
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Correlation analyses were conducted between behavioral and biochemical endpoints, revealing significant relationships among three key measurements evaluated, namely mechanical sensitivity, NK-1 receptor gene expression, and NK-1 receptor affinity (Fig. 9). Both receptor affinity (EC50) and mechanical sensitivity (von Frey threshold) were significantly correlated with NK-1 receptor gene expression (Fig. 9, A and B).
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; Kar et al., 1994; Henry et al., 1999). The variable nature of the results of such studies is especially understandable in light of subsequent studies describing the rapid internalization events associated with agonist-induced activation of the NK-1 receptor (Collins et al., 1992; Grady et al., 1995). This study quantified dynamic changes in NK-1 receptor gene expression combined with concurrent measurement of the function of NK-1 receptors utilizing a ligand-stimulated GTPS35 binding assay performed on sections of lumbar spinal cord, providing valuable new insights into changes in the function and expression of the NK-1 receptor after peripheral inflammation.
Behavioral Impact of Inflammatory Stimuli. Extensive quantification of the hyperalgesia resulting from the inflammatory pain models used in this study was performed; the formalin-evoked hypersensitivity of peripheral tissues (Fu et al., 2000, 2001) is sparsely studied compared with its ability to evoke spontaneous pain-related behaviors (Dubuisson and Dennis, 1977; Hunskaar et al., 1985). As in previous reports, injection of either CFA or formalin produced mechanical or thermal hyperalgesia in the rat (Ma and Woolf, 1996). Plantar thermal and mechanical measurements in formalin-injected animals were confounded because of the overlap of the testing surface and the tissue area injured by the injection. Formalin, although a potent nociceptive activator, also causes irreparable damage to the site of injection due to protein cross-linking. The thermal analgesimeter used in this study allowed testing only of the plantar surface of the paw or tail. Mechanical thresholds measured on the dorsal surface, however, showed formalin-injected rats to be hyperalgesic, but these measurements could not be confirmed through plantar thermal or mechanical testing.
Formalin injection evoked mechanical hyperalgesia on the dorsal surface of the injected hind paw at times when de novo synthesis and receptor affinity were increasing and functional receptor number was decreasing. It is surprising that the most robust hyperalgesia was not associated with increased functional density of NK-1 receptors but rather with their coupling affinity, as described in further detail below. Nonetheless, the temporal pattern of similarities between behavioral sensitization and dynamic changes in NK-1 function suggests that these phenomena are mechanistically linked.
Alterations in Maximal NK-1 Receptor Responsiveness (Emax). At 1 h postformalin, a significant decrease in the Emax of NK-1 stimulated GTPS35 binding was apparent (Fig. 7). This decrease could be attributed to a depletion of available NK-1 receptor binding sites at the surface membrane due to internalization events or to modification of the receptor protein via phosphorylation or interactions with accessory proteins (Collins et al., 1992). It has been previously reported that NK-1 receptors are rapidly internalized after peripheral injection of formalin but are recycled or replaced in the membrane within 90 min postinjection (Wang and Marvizon, 2002). The current results robustly support this finding and may functionally confirm that receptors within the internalized vesicles are either not coupled to G-proteins or are unavailable for ligand binding. However, time course data (Fig. 7) show that, by 6 h, the number of receptors in the membrane functionally coupled to G-proteins is increasing, although the number of coupled receptors on the treated side is still significantly lower than control.
Between 12 and 24 h, the Emax again declined, but this decrease was unlikely due to internalization events, suggesting that a different mechanism is responsible for the decrease in functional receptors seen from 12 to 24 h postformalin. Figure 3 shows a significant increase in mRNA levels for the NK-1 receptor from 2 to 96 h postformalin treatment. Thus, the decrease in the number of functionally coupled NK-1 receptors is not because of down-regulation of NK-1 receptor gene expression. The decrease in Emax could be a result of uncoupling of receptor and G-protein. Inflammation causes an afferent barrage of pharmacological activation, resulting in a flood of activation of intracellular message cascades that could shift the stoichiometry between available receptors and G-proteins. It is possible that NK-1 receptors remaining in the membrane must compete in a depleted pool of G-proteins; thus, the increase in gene expression, in an adaptive fashion, helps overcome this competition by increasing the number of receptors vying for functional coupling to available G-proteins. Ultimately, it appears that the number of functioning NK-1 receptors returns to baseline, an event most likely achieved by elevated levels of NK-1 receptor mRNA and de novo synthesis. The late timing of the return to basal of number of functional receptors may also support the idea that a population of previously non-NK-1-expressing neurons is undergoing a phenotypic switch.
Alterations in NK-1 Receptor Sensitivity (EC50). It is interesting that inflammation also evoked a time-dependent leftward shift in the EC50 of the treated side after formalin (Fig. 5), which was further supported by the time course data (Fig. 8). NK-1 receptors in the ipsilateral lumbar spinal dorsal horn showed a robust decrease in EC50 to 10 to 15% of control levels, which remained reduced as long as 96 h. This leftward shift, representing an increase in receptor coupling affinity, occurs at times when the animal is profoundly hyperalgesic, and the maintenance of increased receptor affinity could precondition the animal to further nociceptive events. Receptors in the contralateral dorsal horn transiently shift to a lower affinity state, but the magnitude of change was not significant. In this regard, NK-1 receptor function may be controlled by mechanisms similar to those regulating NK-1 gene expression, revealing bilateral effects of a unilateral nociceptive stimulus (McCarson, 1999). The mechanisms potentially responsible for producing this phenomenon have been considered previously (McCarson and Krause, 1995). Accordingly, throughout this study, controls were naive animals rather than the contralateral side of the same subject.
Previous publications have described the existence of two distinct receptor populations defined by affinity state. Receptors in the low-affinity state are potentially not functionally coupled to G-proteins, whereas high-affinity receptors are coupled (Kwatra et al., 1993). The GTPS35 binding assay measures only the latter population. Thus, the current results do not reflect a shift from mostly uncoupled to mostly coupled NK-1 receptors but rather an increase in coupling efficiency of the high-affinity receptors after nociceptive activation. Direct quantitation of the relative proportions of high- and low-affinity receptors would require modified binding assays and Scatchard analyses or manipulation of G protein number or availability.
CFA versus Formalin-Evoked Changes in NK-1 Coupling. Perhaps it is surprising that CFA treatment for 4 days evoked a significant decrease in Emax but had no effect on EC50. Previous results have demonstrated that injection of CFA into the hind paw evokes significant increases in NK-1 receptor gene expression in the dorsal horn of the spinal cord ipsilateral to injection (McCarson and Krause, 1994). If alteration of gene expression were the driving force behind changes in receptor affinity, a CFA-evoked shift in EC50 would be anticipated. However, CFA causes a more chronic, less acutely injurious wound than formalin and markedly fewer spontaneous pain-related behaviors. Furthermore, there is a lack of NK-1 receptor internalization upon initial injection of CFA (Honore et al., 1999). These observations, combined with the current results, provide further support for the hypothesis that dynamic alterations in NK-1 receptor functional coupling are driven by mechanisms that result in robust internalization of NK-1 receptors and significant spontaneous pain-related behaviors rather than just development of mechanical or thermal hyperalgesia. NK-1 receptor internalization can, however, be produced if a CFA-inflamed paw is subsequently manipulated (Honore et al., 1999); had this study addressed NK-1 receptor function at times after this type of stimulation, changes in EC50 similar to those evoked by formalin may have been produced.
Relationships among NK-1 Receptor Gene Expression, Functional Coupling, and Nocifensive Behavior. The relationships among changes in NK-1 gene expression, NK-1 receptor affinity in the dorsal horn, and mechanical withdrawal threshold in the paw from 0 to 96 h after formalin were addressed in greater detail using correlation analyses. Figure 9, A and B, shows that both receptor affinity (EC50) and mechanical sensitivity (von Frey threshold) were significantly correlated with NK-1 receptor gene expression, supporting the hypothesis that NK-1 receptor gene expression directly contributes to the plasticity of NK-1 receptor coupling and nociceptive pathway sensitization. These results support previous findings suggesting a correlation between spinal NK-1 immunolabeling and mechanical hypersensitivity after CFA (Goff et al., 1998). Indeed, although these correlations provide enticing speculative insights into mechanisms underlying the role of NK-1 receptors in central sensitization, they do not imply causality.
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Acknowledgements |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: NK-1, neurokinin-1; SP, substance P; CFA, complete Freund's adjuvant; smSP, Sar9Met11(O)2 substance P; PLSD, protected least significant difference; L-733,060, (2S,3S)3-([3,5-bis(trifluoromethyl)phenyl]methoxy)-2-phenylpiperidine; ANOVA, analysis of variance.
Address correspondence to: Dr. Kenneth E. McCarson, University of Kansas Medical Center, Department of Pharmacology, Toxicology, and Therapeutics, Mail Stop 1018, 3901 Rainbow Boulevard, Kansas City, KS 66160-7417. E-mail: kmccarso{at}kumc.edu
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