Introduction

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The neurokinins are a family of endogenous neuropeptides that include substance P (SP), neurokinin A (NKA), and neurokinin B (NKB), agonists at the three known neurokinin receptors (NK1R, NK2R, and NK3R, respectively). These peptides and receptors are present in both the central nervous system and peripheral tissues and mediate a variety of biologic functions, including the transmission of nociceptive information (for review, see Nakanishi, 1991).

There is compelling evidence linking neurokinins and neurokinin receptors to nociceptive signaling. SP and NKA are costored in large, dense-core vesicles in the central terminals of unmyelinated afferent C-fibers that terminate in lamina I, outer lamina II, and lamina V of the spinal dorsal horn (Ogawa et al., 1985). Accordingly, immunoreactivity for NKRs is most dense in the superficial dorsal horn (Zerari et al., 1997, 1998; Honoré et al., 1999). NK1R are activated by noxious, but not by non-noxious stimuli (Honoré et al., 1999). In models of spontaneous pain and hyperalgesia, NK1R, NK3R, preprotachykinin-A (precursor for SP and NKA), and preprotachykinin-B (precursor for NKB) mRNAs are up-regulated in spinal cord (McCarson and Krause, 1994; McCarson, 1999). In rats with selective destruction of spinal NK1R-expressing neurons (by SP-saporin), capsaicin causes a less robust hyperalgesia than in normal rats (Nichols et al., 1999). NK1R antagonists reduce phase II of the formalin test, the development of inflammatory hyperalgesia, and NK1R agonist-induced potentiation of tail-flick latencies and caudally directed scratching and biting (Traub, 1996; Iyengar et al., 1997).

The existence of spinal NK2R has been controversial. Early studies with poorly selective radioligands demonstrated NK2R in the spinal cord (Ninkovic et al., 1984). Subsequent studies with more selective radioligands failed to detect NK2R in the spinal cord (Matuszek et al., 1998). However, this probably relates to the sensitivity of the technique because low levels of NK2R mRNA have been detected in rat spinal cord (Takeda and Krause, 1991). Recently, NK2R have been visualized on astroglial cells in rat spinal cord with the highest concentration in lamina I (Zerari et al., 1998). Behavioral studies supporting the existence of spinal NK2R have shown that intrathecal NK2R antagonists reduce thermal hyperalgesia after burn injury (Lofgren et al., 1999), neuropathic mechanical hyperalgesia (Coudore-Civiale et al., 1998), and both thermal and mechanical allodynia in streptozocin diabetic rats (Coudore-Civiale et al., 2000). In models of visceral pain and hyperalgesia, an NK2R antagonist reduced abdominal contractions induced by intraperitoneal acetic acid (Julia and Bueno, 1997) and responses to rectal distension (Julia et al., 1994).

Less is known of the role of NKB and NK3R activation in pain and hyperalgesia. In the spinal dorsal horn, the majority of NKB is in intrinsic spinal neurons (Ogawa et al., 1985). NK3R (or its mRNA) have been found in the brain and spinal dorsal horn, but not in dorsal root ganglia or other peripheral tissues (McCarson, 1999). In the spinal dorsal horn, NK3R are found on lamina I and II neurons (Yashpal et al., 1990). NK3R in lamina II have also been localized to nerve terminals (Zerari et al., 1997), including SP-containing primary afferent terminals where their activation enhances SP release (Schmid et al., 1998). In cutaneous models of hyperalgesia, NK3R are up-regulated in the dorsal horn (McCarson and Krause, 1994). In acute visceral pain, an NK3R antagonist reduced both the number of abdominal contractions and responses of pelvic nerve afferents to noxious colonic distension (Julia et al., 1999).

Greater than 80% of visceral afferents contain SP (whereas fewer than 25% of cutaneous afferents do; Perry and Lawson, 1998), suggesting that neurokinins are especially important for visceral pain transmission. The present experiments studied the effects of spinal administration of neurokinin receptor antagonists on visceral hyperalgesia by using the model of colorectal distension (CRD). Selective antagonists for each neurokinin receptor were tested alone and in combination in rats with zymosan-induced visceral hyperalgesia (Coutinho et al., 1996). Because it has been well documented that spinal administration of NK1R antagonists can cause motor deficits, the NK2R and NK3R antagonists were tested for motor effects. Preliminary reports of some of these data have appeared in abstract form (Kamp et al., 2000).

	Materials and Methods

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Animals

Adult male Sprague-Dawley rats (400-425 g; Harlan Bioproducts for Science, Indianapolis, IN) were used in all experiments. Rats were housed singly with free access to food and water. All experimental procedures adhered to the International Association for the Study of Pain Research Guidelines and were approved by the Institutional Animal Care and Use Committee at The University of Iowa.

Surgical Preparation

Rats were anesthetized with pentobarbital sodium 50 mg/kg i.p. (Abbott Diagnostics, North Chicago, IL) and electrodes (Teflon-coated stainless steel wire; Cooner Wire Sales, Chatsworth, CA) were sewn into the external oblique abdominal musculature, just above the inguinal ligament, for electromyographic (EMG) recording. At the same time, intrathecal (i.th.) catheters (polyethylene tubing PE-10, 8.5 cm) were placed extending to the lumbar enlargement. The EMG electrodes were subcutaneously guided to the dorsum of the neck and externalized for future access. The wounds were closed in layers with 4-0 silk. After surgery, rats were given 10 ml of 5% dextrose in normal saline (to replace fluids lost during surgery and to provide some nutrition) and allowed to recover for a minimum of 5 days before testing. Any animals with apparent motor defects or that lost significant weight after surgery were excluded. In rats used for motor (rotorod) testing, EMG electrodes were not placed.

Visceral Nociceptive Testing

Behavioral Testing. Contraction of the abdominal musculature during CRD in awake rats was the behavioral response quantified. On the day of testing, a 7-cm-long flaccid latex balloon tied to Tygon tubing was lubricated with Surgilube (E. Fougera and Co., Melville, NY) and inserted intra-anally until the end of the balloon was 1 cm inside the rectum (total insertion distance, 8 cm). The tubing was taped to the base of the tail to prevent displacement and connected to a constant pressure control device (Bioengineering, The University of Iowa, Iowa City, IA) that regulated inflation of the balloon. Materials and methods for CRD and recording and analysis of the EMG signal are fully described elsewhere (Gebhart and Sengupta, 1996). Each distension trial lasted 40 s; EMG activity was quantified during the 10 s before distension (baseline), 20 s during distension, and 10 s after distension.

Experimental Protocol. On the day of testing, five phasic distensions (80 mm Hg, 20 s), at 4-min intervals, were given to establish a baseline response magnitude. Rats were then briefly anesthetized with halothane and either zymosan (1 ml, 25 mg/ml in saline; Sigma, St. Louis, MO) or an equal volume of sterile, preservative-free saline (vehicle) was instilled into the distal colon by using a 7-cm-long, 16-gauge stainless steel feeding needle connected to 1-ml syringe. Three hours after instillation of zymosan or saline, five phasic distensions were repeated (as described above) to evaluate the magnitude of colonic hyperalgesia.

Data Analysis. All experimental groups consist of 5 to 12 rats. The visceromotor response to CRD is represented as percentage of control (% control), where the mean of the prezymosan (naive) responses is defined as 100%. All 20 s of the distension period was used for analysis. The overall effect of any treatment was determined by taking the area under the curve (AUC) of the time-response function for the 4- to 20-min time points, inclusive. Although drug effects were tested for 60 min, inspection of the data revealed that effects of the different drugs tested were reliably maximum during the first 20 min after i.th. drug administration. The AUC was calculated as the sum of the changes in the postdrug response (% control) from the prezymosan (naive) response (100%) plotted against time by using the trapezoidal rule (AUC = response × 16 min). By using this method, a drug that lowers responses to less than baseline (analgesia) will have a negative AUC. If the drug lowers responses to baseline but not further (antihyperalgesia), the AUC is zero. If the drug has no effect or enhances the responses (pronociceptive), the AUC is positive. The AUC data were analyzed by a one-way ANOVA. The Bonferroni correction for multiple comparisons was used if p < 0.05 for the ANOVA. A t test was used to compare the effect of intracolonic saline and zymosan (SigmaStat; Jandel Scientific, San Rafael, CA). Statistical significance is indicated when p < 0.05.

Motor Function Testing

Experimental Protocol. To determine the effect of neurokinin receptor antagonists on motor function, a rotorod test was used. The rotorod apparatus consisted of an elevated drum (6.5 cm in diameter) with a textured surface that rotated at a constant speed of 4.5 rpm. The height of the drum from the floor of the test apparatus was 28 cm. Rats were trained on the rotorod until they could walk continuously for 120 s. Rats that could not meet this criterion were excluded. After training, each rat was loosely restrained in a canvas garden glove and a drug was injected through the chronically implanted i.th. catheter (as in CRD tests described above). Rats were tested 8, 30, and 60 min later. Each test consisted of three opportunities (trials) to remain on the apparatus continuously for 60 s. Animals were returned to their cages between trials and until the next time point.

Data Analysis. Experimental groups consisted of three to nine rats. Because only integer values were recorded for each time point (each rat was successful zero, one, two, or three of three trials), nonparametric analysis of the data was used. Calculation of median and percentile values was done by SigmaPlot (Jandel Scientific). Comparisons were done using the Mann-Whitney U test (a nonparametric, unpaired t test) against vehicle (i.th.) (Minitab for Windows, Version 11.12; Minitab Inc., State College, PA). Statistical significance is indicated when p < 0.05.

Drugs

The drugs used in this study were the NK1R antagonist SR140,333; the NK2R antagonist SR48,968; and the NK3R antagonist SR142,801. Also tested was SR142,806, an enantiomer of SR142,801 with significantly reduced NK3R binding affinity. All were generous gifts from Sonofi Research (Montpellier, France). Stock solutions were prepared by dissolving the drugs in 50% DMSO in sterile, preservative-free saline and then diluted as needed. Drug combinations were prepared by dissolving the NK3R antagonist in the desired concentration of either the NK1R antagonist or the NK2R antagonist. The NK1R/NK2R antagonist combination was prepared by mixing stock solutions containing a single antagonist and diluting to the desired concentration. The lack of effect of vehicle (50% DMSO) was determined in preliminary experiments.

Colonic Inflammation

The myeloperoxidase (MPO) assay was used to quantitate colonic inflammation. Rats were killed by an overdose of pentobarbital and the distal colon removed via laparotomy. The fresh tissue was suspended in hexadecyltrimethylammonium bromide (a detergent), minced with scissors, homogenized/sonicated, and freeze-thawed three times. The tissue suspensions were centrifuged and the supernatant assayed for MPO activity spectrophotometrically by measuring the change in absorbance to 460 nm with time. The color change was accomplished by mixing an aliquot of the supernatant with phosphate buffer containing 0.0005% hydrogen peroxide and O-dianisidine hydrochloride (a pH-sensitive indicator). The greater the rate of conversion of hydrogen peroxide into acid (by MPO), the more intense the color and the greater MPO present in the tissue.

Rats in these experiments received either no treatment (naive), intracolonic saline (3 or 24 h before testing), or intracolonic zymosan (1 ml, 25 mg/ml, 3 or 24 h before testing). A separate group of rats received five distensions (20 s at 80 mm Hg, 4 min apart). Immediately after the fifth distension, these rats received intracolonic zymosan (as described above). Three hours after zymosan, these rats received another set of five distensions. Colons were removed for the MPO assay immediately after the final distension.

The data were analyzed by a one-way ANOVA. The Bonferroni correction for multiple comparisons was used if p < 0.05 for the ANOVA (SigmaStat; Jandel Scientific). Statistical significance is indicated when p < 0.05.

	Results

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Intracolonic Zymosan Produces Hyperalgesia. CRD to 80 mm Hg is a noxious stimulus producing robust contractions of the abdominal musculature, termed the visceromotor response. The magnitude of this response is significantly enhanced after intracolonic administration of zymosan (Coutinho et al., 1996). As seen in Fig. 1, responses to noxious CRD were significantly greater in zymosan-treated rats than in saline-treated rats. In this figure and those that follow, an AUC analysis was performed on responses to CRD between the 4- to 20-min time points. As illustrated, prior intracolonic instillation of zymosan produced a visceral (colonic) hyperalgesia.

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Effect of treatment with intracolonic zymosan on the visceromotor response to noxious CRD. Responses to CRD (80 mm Hg, 20 s) were recorded from naive rats, 3 h after intracolonic zymosan or saline, and at the indicated times after i.th. vehicle (50% DMSO). Data are represented as mean ± S.E.M., calculated as percentage of control, where the response of each rat before zymosan or saline (baseline, b) is defined as 100%. The inset presents the corresponding AUC of the 4- to 20-min times of the above-described time courses. Responses to CRD were significantly enhanced by intracolonic zymosan relative to saline-treated animals (*p < 0.05, t test; n = 10 for both groups).

Motor Function. NK1R antagonists given i.th. produce motor defects in rats (Vaught and Scott, 1987; Traub, 1996), but NK2R and NK3R antagonists have not been similarly studied. We tested the dose-dependent effects of i.th. SR48,968 and SR142,801 on motor function by using the rotorod test. Figure 2 presents the rotorod performance of rats 8 min after i.th. drug (or vehicle) administration. Rats lacking i.th. catheters (naive) rarely fell off the rotorod during the 60-s trials. Vehicle (50% DMSO i.th.) caused some reduction in rotorod performance that was not significantly different from naive rats. Rats receiving 100 µg (i.th.) of either the NK2R antagonist SR48,968 or the NK3R antagonist SR142,801 had significantly decreased performance on the rotorod 8 min after drug administration. A greater dose of the NK2R antagonist SR48,968 (300 µg i.th.) caused flaccid paralysis and also significantly decreased rotorod performance in all rats 8 and 30 min after administration, with recovery seen by 60 min (30- and 60-min data not shown). Lesser doses (30 and 60 µg i.th.) of either the NK2R antagonist SR48,968 or the NK3R antagonist SR142,801 produced no obvious deficits in motor function or degradation of rotorod performance at any time tested. In addition, coadministration of the NK1R antagonist and the NK2R antagonist (3 and 60 µg respectively, i.th.) did not reduce rotorod performance. Doses that significantly affected motor function were not considered analgesic/antihyperalgesic.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Effect of i.th. administration of the NK1R, NK2R, and NK3R antagonists (a) SR140,333, SR48,968, and SR142,801 on motor function. The box plots compare effects of the NKR antagonists on rotorod performance 8 min after i.th. administration. The number of successes (zero, one, two, or three) per three trials was recorded. The thick line represents the median value, the box spans the 25th and 75th percentiles, and the error bars represent the 10th and 90th percentiles. SR48,968 and SR142,801 caused significant motor impairment at doses of 100 µg and higher (i.th.). Coadministration of 3 µg of SR140,333 with 60 µg of SR48,968 produced no significant reduction in rotorod performance [*p < 0.05; Mann-Whitney U test versus i.th. vehicle (50% DMSO); n = 3-9].

Neurokinin Receptor Antagonists Given Singly. To determine the involvement of spinal NK1R in visceral hyperalgesia, i.th. administration of the NK1R antagonist SR140,333 was tested in intracolonic zymosan-treated (hyperalgesic) rats. Figure 3A shows that 10 and 30 µg of SR140,333 reduced responses to noxious CRD. However, obvious motor deficits were observed in animals that received i.th. doses of 6 (data not shown), 10, or 30 µg (e.g., flaccid paralysis, belly-down posture, delayed/absent righting reflex). Animals given a dose of 3 µg of SR140,333 showed no observable motor impairment; however, there was no significant effect on responses to noxious distension at this i.th. dose. Subcutaneous administration of 3 µg of SR140,333 also did not significantly affect responses to CRD in zymosan-treated rats (data not shown). The AUC evaluation presented in Fig. 3B clearly demonstrates that the only doses with a significant effect on responses to CRD were those doses that also produced significant motor effects (m).

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Effect of i.th. administration of the NK1R antagonist SR140,333 on responses to noxious CRD. A, time course of the effect of the NK1R antagonist on zymosan-produced visceral hyperalgesia. Responses to CRD (80 mm Hg, 20 s) were recorded from naive rats, 3 h after intracolonic zymosan (zym), and at the indicated times after i.th. drug or vehicle. Data are represented as mean ± S.E.M., calculated as percentage of control where the response of each rat before zymosan (baseline, b) is defined as 100%. B, corresponding area under the curve of the 4- to 20-min points on the above-described time courses for evaluation of dose-dependent effects. The only doses significantly different from vehicle were 10 and 30 µg (*p < 0.05 versus vehicle, one-way ANOVA with the Bonferroni correction for multiple comparisons; n = 5-10); these doses also produced obvious motor deficits (m).

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Effect of i.th. administration of the NK2R antagonist SR48,968 on responses to noxious CRD. A, time course of the effect of the NK2R antagonist on zymosan-produced visceral hyperalgesia. Responses to CRD (80 mm Hg, 20 s) were recorded from naive rats, 3 h after intracolonic zymosan (z), and at the indicated times after i.th. drug or vehicle. Data are represented as mean ± S.E.M., calculated as percentage of control where the response of each rat before zymosan (baseline, b) is defined as 100%. B, corresponding area under the curve of the 4- to 20-min points on the above-described time courses for evaluation of dose-dependent effects. The only dose significantly different from vehicle was 100 µg (*p < 0.05 versus vehicle, one-way ANOVA with the Bonferroni correction for multiple comparisons; n = 6-10); this dose also produced significant motor effects (m; Fig. 2).

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Effect of i.th. administration of the NK3R antagonist SR142,801 on responses to noxious CRD. A and B, time courses of the effect of the NK3R antagonist in zymosan-treated (hyperalgesic) (A) and saline-treated (nonhyperalgesic) (B) rats. Responses to CRD (80 mm Hg, 20 s) were recorded from naive rats, 3 h after intracolonic zymosan or saline (3 h), and at the indicated times after i.th. drug or vehicle. Data are represented as mean ± S.E.M., calculated as percentage of control where the response of each rat before intracolonic zymosan or saline (baseline, b) is defined as 100%. C, corresponding area under the curve of the 4- to 20-min points on the above-described time courses for evaluation of dose-dependent effects. At the 60-µg dose, the NK3R antagonist significantly reduced responses (versus vehicle, veh) in both hyperalgesic and nonhyperalgesic rats (*p < 0.05 versus vehicle, one-way ANOVA with the Bonferroni correction for multiple comparisons; n = 8-10). Hyperalgesia in zymosan-treated rats is confirmed by the significantly greater AUC (i.th. vehicle) relative to saline-treated rats.

Combinations of Neurokinin Receptor Antagonists. NK1R, NK2R, and NK3R are on different cell populations in the spinal dorsal horn (see Discussion). Presumably then, each receptor could mediate nociception via a separate mechanism; however, this hypothesis is untested. If indeed separate signaling pathways exist, we would expect that combinations of NK1R, NK2R, and NK3R antagonists would be more efficacious than administration of any single antagonist. The highest dose of each antagonist lacking motor effects was chosen for testing. Motor defects were not observed when antagonists were given in combination (Fig. 2).

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Effect of simultaneous i.th. administration of NKR antagonists on responses to noxious CRD in zymosan-treated rats. A, time course of the effect of simultaneous administration of pairs of neurokinin receptor antagonists: the NK1R antagonist (NK1Ra) SR140,333, the NK2R antagonist (NK2Ra) SR48,968, and the NK3R antagonist (NK3Ra) SR142,801. Responses to CRD (80 mm Hg, 20 s) were recorded from naive rats, 3 h after intracolonic zymosan (zym), and at the indicated times after i.th. drugs or vehicle. Data are represented as mean ± S.E.M., calculated as percentage of control where the response of each rat before zymosan (baseline, b) is defined as 100%. B, corresponding area under the curve of the 4- to 20-min points on the above-described time courses. Single administration data from Figs. 3 to 5 are replotted for comparison. Simultaneous administration of the NK1R and NK2R antagonists reduced responses to CRD at doses that had no effect alone. Simultaneous administration of the NK3Ra with either the NK1Ra or the NK2Ra reduced responses to CRD, but this effect was not greater than the effect of the NK3R antagonist alone. (*p < 0.05 versus vehicle; p < 0.05 versus NK1R antagonist alone; one-way ANOVA with the Bonferroni correction for multiple comparisons; n = 7-10).

	Discussion

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Intracolonic Zymosan Models Irritable Bowel Syndrome (IBS). The defining characteristics of IBS are visceral pain accompanied by diarrhea or constipation in the absence of a demonstrable pathology (Thompson et al., 1999). IBS is currently considered a disorder of altered motility and sensation from the small and large intestines. Several studies have shown that IBS patients have decreased visceral pain thresholds and increased areas of referred pain (i.e., visceral hyperalgesia; for review, see Mayer and Gebhart, 1994). This report confirms that intracolonic instillation of zymosan produces a visceral hyperalgesia as determined by increased responses to a noxious intensity of CRD (Coutinho et al., 1996). Because infiltration of neutrophils and inflammation are not associated with intracolonic instillation of zymosan (see Results), the exaggerated responses to CRD model the discomfort and pain that characterize IBS.

NKR Antagonists Cause Motor Impairment. That i.th. NK1R antagonists produce motor deficits in rats has been well documented. For example, the NK1R antagonist [D-Pro2,D-Trp7,9,]-SP produces flaccid paralysis of the hindlimbs (Vaught and Scott, 1987) and the nonpeptide NK1R antagonist CP 96,345 (i.th.) causes paralysis of the hindlimbs, but at greater doses than required for antinociception (Traub, 1996). However, this motor deficit was not NK1R-specific because the stereoisomer (with no NK1R binding affinity) causes equal motor dysfunction (Yamamoto and Yaksh, 1991). Motor deficits caused by i.th. NK2R or NK3R antagonists have not been previously reported in rats. A few published reports mention that no behavioral changes were noted with i.th. administration (Ishizuka et al., 1995; Coudore-Civiale et al., 1998). In mice, subcutaneous administration of the NK2R antagonist SR48,968 decreases rotorod performance (Seguin et al., 1995). In rats, the NK3R agonist senktide (i.th.) causes transient hindlimb flaccidity, decreased time on the rotorod, and thermal hyperalgesia (Linden and Seybold, 1999). However, at the time of maximal hyperalgesia, there is no motor impairment (Linden and Seybold, 1999). The present study found that both the NK2R antagonist SR48,968 and the NK3R antagonist SR142,801 at doses of 100 µg (i.th.) caused motor impairment. These data suggest that spinal NK2R and NK3R may be involved in motor control. However, the antinociceptive and motor effects may be mediated by different receptors, with the motor effects likely to be due to actions of these compounds on calcium channels.

An NK3R Antagonist Is Antinociceptive/Antihyperalgesic. The NK3R antagonist (SR142,801, 60 µg i.th.) significantly reduced responses to CRD in both normal (saline-treated) and hyperalgesic (zymosan-treated) rats, suggesting that the NK3R mediates normal responses to acute visceral pain at the level of the spinal cord. These effects are antinociceptive and are not due to motor impairment. In other models of hyperalgesia, this NK3R (i.th.) antagonist reduced mechanical hyperalgesia in streptozocin-diabetic and mononeuropathic rats (Coudore-Civiale et al., 1998) and blocked NK3R agonist (senktide)-induced thermal hyperalgesia (Linden and Seybold, 1999). In two visceral pain models, systemic administration of this NK3R antagonist reduced both CRD-induced contractions of the abdominal musculature and CRD-induced excitation of pelvic nerve afferents (Julia et al., 1999). Clearly, NK3R modulate normal responses to a noxious stimulus and their activation may be necessary for the maintenance of hyperalgesia.

NKR Antagonists in Combination. The present study, and others, noted a significant attenuation, but incomplete suppression of responses to a noxious stimulus by an NK3R antagonist (to ~70% of baseline). It has been shown that some NK3R are located on the central terminals of primary afferents in the spinal dorsal horn (Schmid et al., 1998). NK3R agonists enhance and antagonists inhibit SP release from these central terminals (Schmid et al., 1998). Because SP is the endogenous ligand for the NK1R, we hypothesized that antagonizing spinal NK1R could enhance NK3R antagonist-induced antinociception. On the other hand, several reports have described an antinociceptive effect of spinal NK3R agonists in behavioral tests (Laneuville et al., 1988; Couture et al., 1993). From these reports, one might hypothesize that the addition of an NK1R antagonist could block NK3R antagonist-induced antinociception.