Nerve-mediated motility of ileal segments isolated from NK1 receptor knockout mice

R. Saban1, N.-B. Nguyen1, M. R. Saban1, N. P. Gerard2, and P. J. Pasricha1

1 The Enteric Neuromuscular Diseases and Pain Laboratory, Division of Gastroenterology and Hepatology, University of Texas Medical Branch, Galveston, Texas 77555-0632; and 2 Ina Sue Pelmutter Laboratory, Children's Hospital, Harvard Medical School, Boston, Massachusetts 02215


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Tachykinins such as substance P (SP) and neurokinin A (NKA) acting on neurokinin (NK) receptors modulate the nonadrenergic noncholinergic (NANC) neurotransmission in the gastrointestinal tract of several species, but the information about the mouse small intestine is scanty. Both SP and NKA induced concentration-dependent contractions of ileal segments isolated from wild-type mice that were blocked by NK1 and NK2 antagonists, respectively. In contrast, segments isolated from NK1 receptor (NK1-R) knockout mice responded only to elevated concentrations of SP. To reveal the inhibitory NANC (iNANC) responses, tissues were pretreated with atropine and guanethidine. Under these conditions, a tetrodotoxin-sensitive relaxation in response to electrical field stimulation (EFS) was observed. NK1-R knockout mice presented a trend toward an increase in iNANC responses, whereas the NK1-R antagonist significantly potentiated iNANC relaxation in tissues isolated from wild-type mice. NG-nitro-L-arginine methyl ester (100 µM) transformed the relaxant response to EFS into a tetrodotoxin-sensitive, frequency-dependent contraction characteristic of an excitatory NANC (eNANC) system. A NK1-R antagonist abolished the contractile responses of the mouse ileum to EFS, whereas a NK2 receptor antagonist had a trend toward reducing EFS-induced contraction. The eNANC component was absent in NK1-R knockout mice. Measurement of SP-like immunoreactivity indicated similar amounts of SP per gram of tissue isolated from wild-type and NK1-R knockout mice, indicating that the observed differences in response to EFS were not due to a differential peptide content. It is concluded that, in the mouse ileum, both NK1 and NK2 receptors modulated the responses to exogenous tachykinins, whereas NK1 is the primary tachykinin receptor involved in both iNANC and eNANC transmission.

neurokinin receptor; sensory nerves; substance P; CP-99994; SR-48968; SR-142801; nitric oxide; nonadregergic noncholinergic system


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE NATURAL TACHYKININS substance P (SP) and neurokinin A (NKA) and their receptors are present throughout the mammalian gastrointestinal tract, where they are found in both intrinsic and extrinsic primary afferent and motoneurons of the myenteric plexus (14). They control both neuronal and neuromuscular transmission within the enteric nervous system (14). According to RIA and immunologic measurements, SP coexists with NKA, and both peptides are coreleased from the enteric nervous system (14). There is considerable evidence to suggest the involvement of tachykinins in the excitatory component of the nonadrenergic noncholinergic (NANC) transmission in the gastrointestinal tract. This has been demonstrated with the use of nonpeptide antagonists of tachykinin receptors in several species, including humans (26). Both neurokinin-1 (NK1) and NK2 receptors have been suggested to modulate excitatory NANC (eNANC) responses. NK1 was found to be the predominant receptor mediating eNANC responses in the circular muscle of the guinea pig colon (25), whereas both NK1 and NK2 receptors mediate the eNANC neuromuscular transmission in the human ileum (26).

Recently, it has been proposed that tachykinins also modulate the inhibitory NANC (iNANC) responses. This suggestion was based on findings describing a coupling between capsaicin-sensitive neurons and lower esophageal sphincter relaxation in the ferret (24). In this species, repeated esophageal acidification causes lower esophageal sphincter relaxation, which is mediated by a peripheral NK1 receptor mechanism (24).

The modulation of gastrointestinal motility by tachykinins also has important pathological and pharmacological implications. There is increasing evidence that tachykinins participate in the hypersecretory, vascular, and immunological disturbances associated with infection and inflammatory bowel disease (15). More recently, it was proposed that part of the mode of action of laxatives involves activation of NK1 and NK2 receptors and a subsequent release of nitric oxide (13).

Recent advances in molecular biology and bioengineering have allowed the development of several knockout mice that may be useful in understanding the role of tachykinins in gastrointestinal motility. Knowledge of the gastrointestinal tachykinin system in the normal mouse is a prerequisite to such an approach but is very limited at the present time (20). The aims of this research were to determine the role of tachykinins in the iNANC and eNANC neurotransmission in the mouse ileum and to define the participation of various NK receptor subtypes in these processes.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. NK1 receptor knockout (NK1-R knockout) and wild-type control mice were generated by Dr. Norma P. Gerard (2), and the colony at University of Texas Medical Branch was genotyped as described previously (2). Male mice weighing 20-29 g were used in this experiment according to the approved animal protocol (University of Texas Medical Branch Animal Care and Use Committee protocol 98-05-033).

Tissues. The animals were anesthetized with ketamine HCl (40 mg/kg im) and xylazine (2.5 mg/kg im), and the ileum was removed. Tissues were placed in a physiological salt solution (PSS, pH 7.4) of the following composition (in mM): 119 NaCl, 1 NaH2PO4, 4.7 KCl, 2.5 CaCl2, 0.5 MgCl2, 25 NaHCO3, and 11 glucose. PSS was maintained at 37°C and aerated continuously with a mixture of 95% O2 and 5% CO2. Ileum segments (~5 mm) were placed as rings on stainless steel stirrups and suspended between two platinum plate electrodes (10 mm apart) in 10-ml water-jacketed tissue baths (37°C) containing PSS. The tissues were attached to force displacement transducers (Grass FT-03; Grass Instruments, Quincy, MA), and changes in tension were displayed on polygraphs (model 79, Grass Instruments). Initial tension on the ileal rings was adjusted at 1 g, which was found to be the optimal tension for this preparation in preliminary experiments. Tissues were allowed to equilibrate for 1 h in PSS containing N-(alpha -rhamnopyranosyloxyhydroxyphosphinyl)-Leu-Trp (phosphoramidon, 1 µM) and (2S)-1-(3-mercapto-2-methylpropionyl)-L-proline (captopril, 1 µM) to inhibit the enzymes neutral endopeptidase and angiotensin-converting enzyme, respectively. During equilibration, PSS was changed and replaced with fresh buffer containing peptidase inhibitors, and tension was readjusted every 15 min.

Preparations were submitted to electrical field stimulation (EFS) using a model S48 stimulator (Grass Instruments) coupled to a stimulus splitter (Stimulus-Splitter III, Med-Lab, Loveland, CO). Frequency-response curves were constructed using trains of 0.16-20 Hz at 10 V and 0.3-ms pulse width for 3 min. EFS was applied after the response to previous stimulation had returned to baseline, which led to an interval between trains of 15-40 min. Contractile and relaxant responses were expressed as percent of the amplitude of the baseline and in some cases as percent of the maximal response obtained with BaCl2 (3 × 10-2 M) added at the end of the experiment.

Potency of agonists. Because the concentration-response curves to tachykinins did not reach a stable plateau at the concentrations used, the potencies of NKA and SP were evaluated at 25% of the maximal responses (EC25) to BaCl2, as described before for other peptides (4, 21). EC25 values were converted to the negative logarithm, and the geometric means of EC25 were determined and expressed as -log molar EC25 (8).

Antagonists. Antagonists were evaluated on the basis of the dose ratio. Apparent dissociation constants (KB) for the antagonists were calculated by the standard equation: KB = [B]/(dose ratio - 1), where [B] is the antagonist concentration. The KB values were converted to negative logarithms and expressed as -log molar KB.

Measurement of SP-like immunoreactivity. To investigate whether different responses to EFS may be attributed to a differential peptide content, SP levels were determined in ileal segments isolated from wild-type and NK1-R knockout mice. SP was extracted from the ileum as described before for other tissues (22). Briefly, segments of ileum (2 cm) were pooled from every three mice, homogenized in 1 ml of 0.2 N perchloric acid, and centrifuged at 10,000 g for 5 min. The supernatant was neutralized by adding an equal volume of 1 M potassium borate (pH 7.4). Samples were centrifuged for 1 min at 10,000 g. The supernatant was collected and stored frozen at -20°C. Specific enzyme immunoassay was used to quantify SP-like immunoreactivity (SP-LI) according to the manufacturer's specifications. The assays were validated in our laboratory for specificity, sensitivity, and reproducibility. SP assay (Cayman Chemical, Ann Arbor, MI) has a sensitivity of 9 pg/ml, intra-assay and interassay variation of <10%, and no cross-reactivity with phosphoramidon or captopril. The specificity of this assay is as follows: SP 100%, SP-(4---11) 97%, SP-(2---1) 93%, SP-(7---11) 30%, eleidosin 12%, neuromedine B 0.04%, SP-(8---11) <0.01%, SP-(1---4) <0.01%, and 2.7% cross-reactivity with NKA (as determined in our laboratory).

Statistical analysis. The statistical analysis of data was performed using Student's paired t-tests where appropriate. Results are expressed as means ± SE. The n values reported refer to the number of animals used for each experiment. In all cases, a value of P < 0.05 was considered indicative of significant difference (1).

Drugs and solutions. The following drugs were purchased from Sigma (St. Louis, MO): atropine sulfate, guanethidine sulfate, NG-nitro-L-arginine methyl ester (L-NAME), HCl papaverine HCl, SP, NKA, L-arginine HCl, tetrodotoxin (TTX), phosphoramidon, and captopril. CP-99994 [(2s-methoxy-benzyl)-(2-phenyl-piperidin-3s-yl)amine] was obtained from Pfizer (Groton, CT), and SR-48968 {(S)-N-methyl-N[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide} and SR-142801 [(S)-(N)-(1-{3-[1-benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl]propyl}-4-phenylpiperidin-4-yl)-N-methylacetamide] were obtained from Sanofi Recherché (Montpellier, France). All solutions were made fresh on the day of the experiment. SR-48968 and SR-142801 were dissolved in 0.1% DMSO, and all other solutions were made in 0.9% sodium chloride.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Tissue SP-LI. Measurement of SP-LI in ileal segments isolated from wild-type and NK1-R knockout mice indicated similar amounts of this peptide per gram of tissue (95 ± 10 and 91 ± 9.0 ng/g tissue, respectively; n = 6, P > 0.05).

Comparison of responses of ileal segments isolated from wild-type and NK1-R knockout mice to exogenously applied tachykinins. We assessed the effect of exogenously applied SP and NKA in the mouse ileum segments by measuring isometric contraction in response to a stepwise increase in peptide concentration. In tissues isolated from wild-type mice, SP and NKA induced a concentration-dependent contraction (Fig. 1). The responses to SP in concentrations above 10-9 M presented a high degree of tachyphylaxis, and the interval between concentrations was increased to 40 min to allow concentration-response curves to be obtained. SP was more potent than NKA (log EC25 of 9.5 ± 0.9 M vs. 8.6 ± 0.8 M; P < 0.05). In addition, the responses induced by SP were inhibited by the NK1 receptor antagonist CP-99994 (KB = 7.3 ± 0.7), whereas the contractions induced by NKA were inhibited by the NK2 receptor antagonist SR-48968 (KB = 7.5 ± 0.6). In contrast, ileal segments isolated from NK1-R knockout mice responded only to high concentrations of SP (Fig. 1A), and the concentration-responses curve to NKA (log EC25 = 7.7 ± 0.9) presented a significant rightward shift compared with those obtained in tissues isolated from wild-type mice (Fig. 1B). The responses to NKA observed in NK1-R knockout mice were significantly reduced by the NK2 receptor antagonist (SR-48968, 1 µM; Fig. 1B).



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Fig. 1.   A: concentration-response curves to substance P (SP) obtained in ileal segments isolated from neurokinin 1 receptor (NK1-R) knockout (KO) and wild-type (WT) mice. Tissues were pretreated with atropine (1 µM) and guanethidine (1 µM) 1 h before dose-response curves were obtained. Responses are expressed as percent of maximal responses to BaCl2, which were 2.9 ± 0.3 and 2.8 ± 0.2 g in tissues isolated from wild-type and NK1-R knockout mice, respectively (P > 0.05). Values are means ± SE (n = 6). * P < 0.05 between wild-type and NK1-R knockout mice. B: effect of SR-48968 on concentration-response curves to neurokinin A (NKA) obtained in ileal segments isolated from NK1-R knockout and wild-type mice. Tissues were pretreated with atropine (1 µM) and guanethidine (1 µM) 1 h before incubation with SR-48968 (1 µM) for 90 min. DMSO (0.01%) was used as vehicle control for SR-48968. Responses are expressed as percent of maximal responses to BaCl2, which were 2.9 ± 0.3 g and 2.8 ± 0.2 g in tissues isolated from wild-type and NK1-R knockout mice, respectively (P > 0.05). Values are means ± SE (n = 6). * P < 0.05 between tissues treated with SR-48968 and DMSO.

Characterization of the iNANC responses of ileal segments isolated from wild-type and NK1-R knockout mice. To reveal the iNANC responses, tissues were pretreated with atropine and guanethidine (both at 1 µM concentrations). Under these conditions, lower frequencies of EFS (0.16-1.3 Hz) induced a frequency-dependent relaxation of ileal segments, and termination of the stimulation produced an off or rebound contraction as illustrated in Fig. 2A. Similar responses were obtained at this low frequency in tissues isolated from both wild-type (Fig. 2A) and NK1-R knockout mice (Fig. 2B). At higher frequencies (2.6-10 Hz), the ileum isolated from wild-type mice presented a biphasic response composed of a relaxation (phase I) followed by a gradual recovery of motility (phase II), as illustrated in Fig. 2C. In contrast, segments isolated from knockout mice failed to present phase II (Fig. 2D). In addition, the magnitude of the off contraction after 10 Hz of stimulation was reduced in tissues isolated from NK1-R knockout compared with wild-type mice (42 ± 10% vs. 81 ± 10% of maximal responses to BaCl2; P < 0.05). The iNANC responses of the mouse ileum to EFS are summarized in Fig. 2E. In separate experiments, it was determined that all responses to EFS were abolished by previous incubation with TTX (1 µM, 1 h; data not shown).






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Fig. 2.   Representative polygraph tracings of responses of isolated mouse ileum to low and high frequencies of electrical field stimulation (EFS) under inhibitory nonadrenergic noncholenergic (iNANC) conditions. Results were obtained in presence of atropine and guanethidine (both 1 µM for 1 h; see METHODS). A and C are from tissues isolated from 2 wild-type mice, and B and D are from tissues isolated from 2 NK1-R knockout mice. Tracings are actual representatives of polygraph tracings of ileum segments isolated from 12 different mice. A: responses of ileal segments isolated from wild-type mice to low-frequency stimulation (1.3 Hz, 10 V, 0.3 ms). B: responses of ileal segments isolated from NK1-R knockout mice to low-frequency stimulation (1.3 Hz, 10 V, 0.3 ms). C: biphasic responses (phases I and II) of ileal segments isolated from wild-type mice to high-frequency stimulation (5.2 Hz, 10 V, 0.3 ms). D: monophasic responses of ileal segments isolated from NK1-R knockout mice to high-frequency stimulation (5.2 Hz, 10 V, 0.3 ms). E: average (mean ± SE) of peak relaxant responses (phase I) to EFS obtained in ileal segments isolated from wild-type (n = 6) and NK1-R knockout mice (n = 6). Relaxant responses were calculated as percent of baseline amplitude [1.0 ± 0.03 and 0.98 ± 0.03 g in tissues isolated from wild-type (WT) and NK1-R knockout (KO) mice, respectively (P > 0.05)].

Effect of NK receptor antagonists on the iNANC response in wild-type mice. We examined the effect of NK receptor antagonists on iNANC responses of ileal segments isolated from wild-type mice. The NK1 receptor antagonist CP-99994 (10 µM) increased the relaxant responses of the mouse ileum to EFS and transformed the biphasic response into a monophasic relaxation as illustrated in Fig. 3A. In contrast, SR-48968 (10 µM) and SR-142801 (10 µM), NK2 and NK3 receptor antagonists, respectively, did not significantly alter the iNANC component (Fig. 3B).



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Fig. 3.   Effect of NK receptor antagonists on iNANC responses obtained in ileal segments isolated from wild-type mice. Responses were obtained under iNANC conditions (atropine and guanethidine, both 1 µM for 1 h; see METHODS). A: representative polygraph tracing of effect of NK1 receptor antagonist CP-99994 (10 µM, 1 h) on responses of isolated mouse ileum to EFS (5.2 Hz, 10 V, 0.3 ms). Tracing is actual representative of ileum segments isolated from 6 different mice. B: frequency-response curves of isolated mouse ileum to EFS (0.64-10 Hz, 10 V, 0.3 ms) in presence of atropine and guanethidine (both 1 µM) and NK receptor antagonists (all at 10 µM) CP-99994 (60 min), SR-48968 (90 min), and SR-142801 (90 min). DMSO (0.01%) was used as vehicle control for SR-48968 and SR-142801. Relaxant responses were calculated as percent of baseline amplitude [1.0 ± 0.05 and 1.0 ± 0.03 g in tissues isolated from wild-type and NK1-R knockout mice, respectively (P > 0.05)]. Values are means ± SE (n = 6).

Characterization of the eNANC responses of ileal segments isolated from wild-type mice and NK1-R knockout mice. In the presence of guanethine and atropine (both at 1 µM concentrations), the addition of the nitric oxide synthase inhibitor L-NAME (100 µM) to the tissue bath induced a slowly developing contraction of the mouse ileum. This contraction reached a stable plateau in 10 min and increased the tonus 29 ± 8% of the maximal response to BaCl2 (3 × 10-2 M) added at the end of the experiment. L-NAME transformed the relaxant response to EFS (represented in Fig. 2, A and C) into a TTX-sensitive, frequency-dependent contraction (Fig. 4A). Lower concentrations of L-NAME (1-10 µM) failed to inhibit EFS-induced relaxation (data not shown). To confirm that the effect observed with 100 µM L-NAME was due to inhibition of nitric oxide synthase, additional experiments were performed using excess L-arginine (1 mM), which completely reversed the effect of L-NAME on the responses of isolated ileum to EFS (data not shown). The eNANC component of the response to EFS was determined in ileal segments isolated from NK1-R knockout mice and compared with wild-type control mice. The eNANC component of the response to EFS was absent in NK1-R knockout mice. These results are summarized in Fig. 4E.






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Fig. 4.   Excitatory NANC (eNANC) responses of ileal segments isolated from wild-type and NK1-R knockout mice. Responses to EFS (5.2 Hz, 10 V, 0.3 ms) were obtained in presence of atropine (1 µM), guanethidine (1 µM), and L-NAME (100 µM) as described in METHODS. Tracings are actual representatives of polygraph tracings of ileum segments isolated from 24 different mice. A: eNANC responses of ileal segments isolated from wild-type mice. B: representative polygraph tracing of effect of NK1 receptor antagonist CP-99994 (10 µM, 60 min) on eNANC responses of ileal segments isolated from wild-type mice. C: frequency-response curves (means ± SE) obtained in tissues isolated from wild-type mice in absence (saline, n = 6) and in presence of NK1 receptor antagonist CP-99994 (10 µM, 60 min, n = 6). Relaxant responses were calculated as percent of baseline amplitude [1.0 ± 0.03 and 1.0 ± 0.03 g in tissues isolated from wild-type and NK1-R knockout mice, respectively (P > 0.05)]. D: frequency-response curves (means ± SE) obtained in tissues isolated from wild-type mice in absence (DMSO, n = 6) and in presence of NK2 receptor antagonist SR-48968 (10 µM, 90 min, n = 6). Relaxant responses were calculated as percent of baseline amplitude [1.0 ± 0.03 and 1.0 ± 0.03 g in tissues isolated from wild-type and NK1-R knockout mice, respectively (P > 0.05)]. E: frequency-response curves (means ± SE) representing eNANC responses obtained in tissue isolated from wild-type (n = 6) and NK1-R knockout mice (NK1-R KO, n = 6); * P < 0.05 between groups. Relaxant responses were calculated as percent of baseline amplitude [0.99 ± 0.05 and 0.98 ± 0.06 g in tissues isolated from wild-type and NK1-R knockout mice, respectively (P > 0.05)].

Effect of NK receptor antagonists on the eNANC response in wild-type mice. In the presence of guanethidine, atropine, and L-NAME, the NK1 receptor antagonist CP-99994 (10 µM) abolished the contractile responses of the mouse ileum to EFS. These results are illustrated in Fig. 4B and summarized in Fig. 4C. The NK2 receptor antagonist SR-48968 (10 µM, 90 min) had a trend toward reducing EFS-induced contraction. However, this reduction was not statistically significant (Fig. 4D). In addition, the NK3 receptor antagonist SR-142801 (10 µM, 90 min) did not alter the ileal eNANC responses to EFS (data not shown).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we have first shown that both NK1 and NK2 receptors mediate the response to exogenously applied tachykinins in ileal segments isolated from wild-type mice. However, the concentration-response curve to SP was obtained only when the time between concentrations was increased to 40 min to avoid tachyphylaxis. Further experiments are necessary to confirm whether the observed tachyphylaxis to SP may indicate rapid endocytosis of the NK1 receptor as has been suggested in enteric neurons (5, 11). Within these limitations, SP was more potent than NKA in inducing contractions at the low doses used in the experiments. At these doses, SP and NKA acted almost exclusively via their preferred receptors, as complete antagonism of their effects was obtained by NK1 and NK2 receptor antagonists, respectively (18). Indeed, SR-48968 blocked NKA responses with an apparent KB of 7.5, and CP-99994 blocked the responses to SP with an apparent KB of 7.3. The latter values are similar to those described for CP-99994 in the mouse stomach (22). The presence of both receptors in the mouse gastrointestinal tract is in accordance with binding experiments indicating the presence of both NK1 and NK2 receptors in other species, including rats and humans (6, 9, 23).

It was also clear from the experiments in the knockout mice that, at higher doses, SP can act on NK2 receptors and that the effects of NKA also include a component mediated by NK1 receptors. It has previously been shown that NKA can bind to NK1 receptors (12); however, whether this results in receptor activation is not clear. Our results suggest that NKA may have a functional effect on NK1 receptors as indicated by the rightward shift of its dose-response curve in the knockout mice when compared with the results in the wild-type mice (Fig. 1B). Another explanation for this shift is that part of the response to NKA in wild-type mice may be due to a release of SP and therefore to action on NK1 receptors, whereas in the NK1-R knockout mice, the response to NKA is solely due to action on NK2 receptors. Supporting this hypothesis is the differential effect of SR-48968 in tissues obtained from wild-type and NK1-R knockout mice (Fig. 1B). A role for acetylcholine and norepinephrine in this response was ruled out because the experiments were conducted in the presence of guanethidine and atropine. These experiments, however, did not exclude the participation of prostaglandins and other mediators.

Our results also indicate a nitrinergically mediated iNANC component of responses to EFS in the mouse ileum, as in other species, including humans (26). There was no difference in the magnitude of the TTX-sensitive relaxant responses between wild-type and knockout mice. Although individual results indicated a trend toward tissues isolated from NK1-R knockout mice presenting an increased relaxation in response to EFS (Fig. 2, C and D), the variability of the experiment was such that the differences between averages did not reach statistical significance (Fig. 2E). However, ileal segments from NK1-R knockout mice present a monophasic relaxation compared with a biphasic response obtained in tissues from wild-type mice. The NK1 receptor antagonist CP-99994 but not NK2 and NK3 antagonists SR-48968 and SR-142801, respectively, also enhanced the relaxation induced by EFS, possibly by reducing the contractile component of the response to EFS. In the presence of L-NAME, EFS induced a frequency-dependent contraction that was sensitive to TTX, indicating its neuronal nature. In the presence of L-NAME, NK1 but not NK2 or NK3 receptor antagonism abolished this eNANC effect. The NK2 and NK3 receptor antagonists had a trend, although not statistically significant, toward inhibiting this response. However, a limitation of the use of both SR-48498 and SR-142801 should be pointed out. These compounds require solution in DMSO, which by itself significantly reduced eNANC contractions in response to EFS when compared with tissue treated with saline (Fig. 4, D and F).

To further confirm the importance of NK1 receptors on the eNANC, we conducted additional experiments using ileal segments isolated from NK1-R knockout mice. Ileal segments isolated from NK1-R knockout mice failed to present contraction with EFS, indicating the predominant role of this receptor subtype in the eNANC system. The differences observed between wild-type and knockout mice were not due to a different tissue concentration of SP because our results indicated similar levels of the peptide in ileal segments isolated from both types of mice.

Other authors have suggested the participation of both NK1 and NK2 receptors on the eNANC. Lecci et al. (16), using a model of colonic propulsion in anesthetized guinea pigs, indicated that both NK1 and NK2 receptors play a fundamental role as eNANC neurotransmitters. Differences in the species of animal, type of muscle (circular vs. longitudinal), or type of organ may provide explanations for these discrepancies. Thus NK1 and NK2 receptors are both present in the circular muscle of the guinea pig colon (6, 17, 19), whereas only NK1 receptors seem to be present in the longitudinal muscle layer (3, 10). Alternative explanations include the suggestion by Maggi and collaborators (24, 26) that the recruitment of specific receptor populations may differ depending on the duration of the stimuli. To avoid this problem, we used a fixed duration of stimulus (0.3 ms) and analyzed the entire frequency-response curve.

How then do we explain the fact that, whereas the mouse ileum contains functional NK1 as well as NK2 receptors, the response to nerve stimulation (which should release both NKA and SP) is completely abolished in the NK1-R knockout mice? A possible explanation for this apparent discrepancy is that NK1 receptors are the ones directly innervated, whereas NK2 receptors are mainly found in extrasynaptic sites in gastrointestinal smooth muscle, as suggested by Grady et al. (10). These authors found NK1 receptors in the myenteric submucosal neurons and in interstitial cells of Cajal, whereas NK2 receptors were localized in the muscularis mucosa of the small intestine and colon. Although the hypothesis of a differential distribution of receptors is very attractive and may explain the singular participation of NK1 receptors in the NANC system, additional experiments are necessary to confirm whether the same is true for the mouse gastrointestinal tract.

In summary, the results obtained with the isolated mouse ileum indicate that both NK1 and NK2 receptors modulate the responses of the mouse ileum to exogenously applied tachykinins and that both SP and NKA have effects on the NK1 receptor. However, in the presence of atropine and guanethidine, the responses induced by nerve stimulation are modulated primarily by NK1 receptors. Together our results indicate that NK1 is the primary tachykinin receptor involved in eNANC transmission in the mouse. As has been demonstrated, various gastrointestinal disorders are associated with distinct changes in the tachykinin system associated with infection (7) and inflammatory bowel disease (15). Therefore, it is conceivable that tachykinin NK1 antagonists could be exploited as antidiarrheal, anti-inflammatory, and antinociceptive drugs.


    ACKNOWLEDGEMENTS

We gratefully acknowledge Dr. Douglas W. P. Hay, SmithKline Beecham Pharmaceuticals (King of Prussia, PA) for the advice regarding NK3 receptor antagonists. We thank Linda A. Hardeman for typing, proofreading, and editing the manuscript.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: R. Saban, The Enteric Neuromuscular Diseases and Pain Laboratory, Division of Gastroenterology and Hepatology, Dept. of Internal Medicine, Univ. of Texas Medical Center, 1108 The Strand, Rm. 219, Galveston, TX 77555-0632 (E-mail: risaban{at}utmb.edu).

Received 5 May 1999; accepted in final form 13 August 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bernard, R. (Editor). Fundamentals of Biostatistics. Boston, MA: PWS, 1990, p. 442-450.

2.   Bozic, C. R., B. Lu, U. E. Höpken, G. Gerard, and N. P. Gerard. Neurogenic amplification of immune complex inflammation. Science 273: 1722-1725, 1996[Abstract/Free Full Text].

3.   Briejer, M. R., L. M. A. Akkermans, A. L. Meulemans, R. A. Lefebvre, and J. J. A. J. Schuurkes. Substance P-induced contraction of the guinea-pig proximal colon through stimulation of post-junctional tachykinin NK1 receptors. Eur. J. Pharmacol. 250: 181-183, 1993[Medline].

4.  Buckner, C. K., S. V. Guanekar, J. S. Kays, R. D. Krell, R. I. Fishleder, J. A. Will, and J. A. Vann. Pharmacological studies of tachykinin receptors mediating contraction of isolated airway smooth muscle. Ann. NY Acad. Sci. 340-358, 1994.

5.   Bunnett, N. W., M. Bouvier, and A. De Blasi. Peptide G protein-coupled receptors meet at Erice. Trends Pharmacol. Sci. 19: 343-346, 1998[Medline].

6.   Burcher, E., S. H. Buch, W. Lovemberg, and T. L. O'Donohue. Characterization and autoradiographic localization of multiple tachykinin binding sites in gastrointestinal tract and bladder. J. Pharmacol. Exp. Ther. 236: 819-831, 1986[Abstract].

7.   Castagliuolo, I., M. Riegler, A. Pasha, S. Nikulasson, B. Lu, C. Gerard, N. P. Gerard, and C. Pothoulakis. Neurokinin-1 (NK-1) receptor is required in clostridium difficile-induced enteritis. J. Clin. Invest. 101: 1547-1550, 1998[Abstract/Free Full Text].

8.   Fleming, W. W., D. P. Westfall, I. S. De La Lande, and L. B. Jellet. Log-normal distribution of equieffective doses of norepinephrine and acetylcholine in several tissues. J. Pharmacol. Exp. Ther. 181: 339-345, 1972[Medline].

9.   Gates, T., R. P. Zimmerman, C. R. Mantyh, S. R. Vigna, J. E. Maggio, M. L. Welton, E. P. Passaro, Jr., and P. W. Mantyh. Substance P and substance K receptor binding sites in the human gastrointestinal tract: localization by autoradiography. Peptides 9: 1207-1219, 1988[Medline].

10.   Grady, E. F., P. Baluk, S. Bohm, P. D. Gamp, H. Wong, D. G. Payan, J. Ansel, A. L. Portbury, J. B. Furness, D. M. McDonald, and N. W. Bunnett. Characterization of antisera specific to NK1, NK2, and NK3 neurokinin receptors and their utilization in the rat gastrointestinal tract. J. Neurosci. 16: 6975-6986, 1996[Abstract/Free Full Text].

11.   Grady, E. F., P. D. Gamp, E. Jones, P. Baluk, D. M. McDonald, D. G. Payan, and N. W. Bunnett. Endocytosis and recycling of neurokinin 1 receptors in enteric neurons. Neuroscience 75: 1239-1254, 1996[Medline].

12.   Hastrup, H., and T. W. Schwartz. Septide and neurokinin A are high-affinity ligands on the NK-1 receptor: evidence from homologous versus heterologous binding analysis. FEBS Lett. 399: 264-266, 1996[Medline].

13.   Izzo, A. A., T. S. Gaginella, N. Mascolo, and F. Capasso. Recent findings on the mode of action of platelet activating factor and nitric oxide. Trends Pharmacol. Sci. 19: 403-405, 1998[Medline].

14.   Holzer, P., and U. Holzer-Petsche. Tachykinins in the gut. Part I. Expression, release and motor function. Pharmacol. Ther. 73: 173-217, 1997[Medline]

15.   Holzer, P., and U. Holzer-Petsche. Tachykinins in the gut. Part II. Roles in neural excitation, secretion and inflammation. Pharmacol. Ther. 73: 219-263, 1997[Medline].

16.   Lecci, A., S. Giuliani, M. Tramontana, R. D. Giorgio, and C. A. Maggi. The role of tachykinin NK1 and NK2 receptors in atropine-resistant colonic propulsion in anaesthetized guinea-pigs. Br. J. Pharmacol. 4: 27-34, 1998.

17.   Maggi, C. A., R. M. Catalioto, M. Criscuoli, S. Giuliani, A. Lecci, A. Lippi, S. Meini, R. Patacchini, A. R. Renzetti, P. Santicioli, M. Tramontana, and G. A. Zagorodnyuk. Tachykinin receptors and intestinal motility. Can. J. Physiol. Pharmacol. 75: 696-703, 1997[Medline]

18.   Maggi, C. A., and T. W. Schwartz. The dual nature of the tachykinin NK1 receptor. Trends Pharmacol. Sci. 18: 351-355, 1997[Medline].

19.   Maggi, C. A., V. Zagorodnyuk, and S. Giuliani. Specialization of tachykinin NK1 and NK2 receptors in producing fast and slow atropine-resistant neurotransmission to the circular muscle of the guinea-pig colon. Neuroscience 63: 1137-1152, 1994[Medline].

20.   Nsa Allogho, S., X. K. Ngugyen-Le, F. Gobeil, L. H. Pheng, and D. Regoli. Neurokinin receptors (NK1, NK2) in the mouse: a pharmacological study. Can. J. Physiol. Pharmacol. 75: 552-557, 1997[Medline].

21.   Saban, R., E. C. Dick, R. I. Fishleder, and C. K. Buckner. Enhancement by parainfluenza-3 infection of contractile responses to substance P and capsaicin in airway smooth muscle from the guinea pig. Am. Rev. Respir. Dis. 136: 586-591, 1987[Medline].

22.   Saban, R., J. Franz, and D. E. Bjorling. Spontaneous release of substance P (SP) and bradykinin (BK) by isolated guinea pig bladder. Br. J. Urol. 79: 516-524, 1997[Medline].

23.   Santicioli, P., S. Giuliani, R. Patacchini, M. Tramontana, M. Criscuoli, and C. A. Maggi. MEN 11420, a potent and selective tachykinin NK2 receptor antagonist in the guinea-pig and human colon. Naunyn Schmiedebergs Arch. Pharmacol. 356: 678-688, 1997[Medline].

24.   Smid, S. D., P. A. Lynn, R. Templeman, and L. A. Blackshaw. Activation of non-adrenergic non-cholinergic inhibitory pathways by endogenous and exogenous tachykinins in the ferret lower esophageal sphincter. Neurogastroenterol. Motil. 10: 149-156, 1998[Medline].

25.   Zagorodnyuk, V., P. Santicioli, and C. A. Maggi. Tachykinin NK1 but not NK2 receptors mediate non-cholinergic excitatory junction potentials in the circular muscle of guinea-pig colon. Br. J. Pharmacol. 110: 705-803, 1993.

26.   Zagorodnyuk, V., P. Santicioli, D. Turini, and C. A. Maggi. Tachykinin NK1 and NK2 receptors mediate non-adrenergic non-cholinergic excitatory neuromuscular transmission in the human ileum. Neuropeptides 31: 265-271, 1997[Medline].


Am J Physiol Gastroint Liver Physiol 277(6):G1173-G1179
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