Physiological role of neuropeptide Y in the regulation of the ascending phase of the peristaltic reflex
John R. Grider and
Lea E. Langdon
Department of Physiology and Medicine, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia 23298
Submitted 18 February 2003
; accepted in final form 26 July 2003
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ABSTRACT
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The physiological role of neuropeptide Y (NPY) and of specific NPY receptors in regulating the intestinal peristaltic reflex was examined in three-compartment flat-sheet preparations of rat colon. Graded muscle stretch or mucosal stimulation applied to the central compartment inhibited NPY release in the orad compartment where ascending contraction was measured. NPY and the Y1-receptor agonist [Leu31, Pro34]NPY inhibited, whereas the selective Y1-receptor antagonist BIBP 3226 augmented ascending contraction and substance P (SP) release in the orad compartment induced by muscle stretch or mucosal stimulation. Neither agonist nor antagonist had any effect on descending relaxation or VIP release in the caudad compartment. The Y2-receptor agonist NPY13-36 and antagonist BIIE 0246 had no effect on peptide release or mechanical response. The results indicate that suppression of a tonic inhibitory influence of NPY neurons on excitatory neurotransmitter release contributes substantially to the orad contractile phase of the peristaltic reflex. The effect of NPY on neurotransmitter release is mediated by Y1 receptors.
enteric nervous system; tachykinins; NPY receptors; colon
NEUROPEPTIDE Y (NPY) belongs to a family of closely related tyrosine (Y)-rich peptides that includes the hormonal peptides peptide YY (PYY) and pancreatic polypeptide (PP). All three peptides are 36 amino acids long and possess a characteristic tertiary structure consisting of a U-shaped polyproline helix (PP-fold) (12, 25). PYY and PP are primarily synthesized and released from endocrine cells of the gut and pancreas, respectively, whereas NPY is exclusively located in central and peripheral neurons, including neurons of the myenteric and submucosal plexi of the enteric nervous system. In most regions of the gut, NPY-containing neurons project within the myenteric plexus and into the circular and longitudinal muscle layers. In most species, including rat, guinea pig, mouse, and human, NPY is colocalized with VIP or nitric oxide (NO) synthase in 50-70% of enteric neurons, particularly in the colon (2, 9, 21, 27, 30).
The function of NPY upon release from myenteric neurons is unknown. The pharmacological effects of NPY applied exogenously are variable: some effects are direct, and others involve stimulation or inhibition of excitatory neurotransmitter release (3, 4, 8, 9, 17, 23-26, 28, 29, 31). These disparate effects depend on the type of receptor expressed at various neural or muscular locations. Five distinct NPY receptors (Y1, Y2, Y4, Y5, and Y6) have now been cloned and characterized pharmacologically with respect to their affinities for the endogenous ligands, NPY, PYY, and PP (10, 25). Two agonists, the Pro34-substituted [Leu31, Pro34]NPY, and the COOH-terminal fragments of NPY or PYY (NPY13-36), which interact preferentially with Y1 and Y2 receptors, respectively, and the nonpeptide antagonists BIBP 3226 and BIIE 0246, which exhibit high affinity for Y1 and Y2 receptors, respectively, have been used to facilitate the analysis of receptor types in native tissues (6-8, 12, 25, 32). Pharmacological and radioligand binding studies suggest that NPY interacts with Y1 receptors on enteric neurons in various mammalian species and with Y1 and Y2 receptors on smooth muscle cells of the circular muscle layer in several species including the rabbit, guinea pig, rat, and human (23-25, 28, 29). Interaction with Y2 receptors mediates inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ release and contraction, whereas interaction with both Y1 and Y2 receptors mediates inhibition of adenylyl cyclase activity (26). Studies by Ferrier et al. (11) using RT-PCR in nerve/muscle segments of proximal rat colon have identified the expression of Y1 and Y4 receptors that appear to be located on enteric neurons. Similarly, immunohistochemical studies have demonstrated the presence of Y1 receptors on cell bodies and nerve fibers of rat intestinal myenteric but not submucosal plexus neurons (24). These studies also suggest that the location of these Y1 receptors is likely to be a presynaptic rather than postsynaptic location. Feletou et al. (10) identified expression of Y2 receptors also that may be located more distally in rat colon, most likely on smooth muscle cells. The latter finding is consistent with the pharmacological results in rat colonic strips (29) and isolated smooth muscle cells from guinea pig, rabbit, and human (26).
The present study attempts to identify a physiological role for NPY and its receptors in the regulation of neuromuscular function of the gut, with particular emphasis on the regulation of intestinal peristaltic activity. Two earlier studies suggested that NPY might play a role in regulating intestinal peristaltic activity. Holzer et al. (17) showed that exogenous application of NPY to an isolated segment of guinea pig ileum inhibited ascending contraction of circular muscle initiated by aboral balloon distension. A subsequent study in guinea pig colon by Krantis and Harding (20) showed that the hormonal peptide PYY, which exhibits a pharmacological profile similar to that of NPY, inhibited peristaltic activity as measured by the rate of propulsion of fecal pellets. In the present study, we have measured the release of NPY during the ascending and descending phases of the peristaltic reflex in the rat colon and used selective Y1 and Y2 receptor agonists and antagonists to identify the role of NPY and NPY receptors in the regulation of the reflex. NPY release decreased during the ascending phase of the reflex. The Y1-receptor antagonist BIBP 3226 augmented ascending contraction and substance P (SP) release, whereas the Y1 agonist [Leu31, Pro34]NPY had the reverse effect. Y1- and Y2-receptor agonists or antagonists had no effect on descending relaxation. The results suggest that NPY exerts a tonic inhibitory influence on excitatory neurotransmitter release. A decrease in this influence during the ascending phase is reflected by an increase in SP release and muscle contraction.
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METHODS
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Measurement of peristaltic reflex in compartmented flat-sheet segments of rat colon. The peristaltic reflex was measured in a three-compartment, flat-sheet preparation of rat colon as described in detail previously (15). A 5- to 7-cm segment of mid- to distal colon was opened along the mesenteric attachment and pinned, mucosal side up, to the Sylgard base of an organ bath such that the in situ dimensions were maintained without applying additional stretch in the circular or longitudinal direction. The segment was divided into three compartments by vertical partitions sealed with vacuum grease, and 2 ml Krebs-bicarbonate medium were added to each compartment. The composition of the medium was (in mM) 118 NaCl, 4.8 KCl, 1.2 KH2PO4, 2.5 CaCl2, 1.2 MgSO4, 25 NaH2CO3, and 11 glucose, to which was added 0.1% bovine serum albumin, 10 µM amastatin, and 1 µM phosphoramidon. The peristaltic reflex was initiated by stroking the mucosa with a fine brush (2-8 strokes at a rate of 1 stroke/s) and by muscle stretch applied in the radial direction with a hook and pulley assembly (2-10 g for 30 s each). Ascending contraction of circular muscle was measured in the orad peripheral compartment, and descending relaxation was measured in the caudad peripheral compartment using force-displacement transducers attached to the muscle layers. For measurement of transmitter release, muscle stretch or mucosal stimuli was applied to the central compartment five times each during a 15-min period; the medium from the central and both peripheral compartments was collected and frozen for subsequent radioimmunoassay. At the end of each experiment, the colonic segment was cut into three sections corresponding to the tissue in the three compartments, and each section was blotted dry and weighed. The tissue wet weight was then used in the normalization of the amount of neuropeptide released into each compartment during the peristaltic reflex. The mean wet weight of tissue was similar in each compartment, and the overall mean wet weight of tissue in all compartments was 147 ± 8 mg. Experiments were repeated in the presence of 1 µM NPY and various Y1 and Y2 agonists and antagonists. In experiments designed to examine the role of the Y1 receptor in the noncholinergic component of the peristaltic reflex, the effects of the Y1 agonist and antagonist were reexamined in the presence of the muscarinic receptor antagonist atropine (1 µM).
Measurement of SP, VIP, and NPY. SP was measured as described previously using antibody RAS 7451 (13, 15). The limit of detection of the assay was 3 fmol/ml, and the IC50 was 12 ± 4 fmol/ml of original sample. The concentration of SP in the samples ranged from 7 to 52 fmol/ml. The antibody reacts fully with SP but does not cross-react with NPY, neurokinin A (NKA), neurokinin B, somatostatin, VIP, or [Met]enkephalin. VIP was measured as described previously using antibody RAS 7161 (14, 15). The limit of detection of the assay was 3 fmol/ml, and the IC50 was 22 ± 8 fmol/ml of original sample. The concentration of VIP in the samples ranged from 9 to 63 fmol/ml. The antibody reacts fully with SP but does not cross-react with VIP, NPY, pituitary adenylate cyclase activating peptide (PACAP), peptide histidine isoleucine (PHI), secretin, glucagon, somatostatin, SP, or [Met]-enkephalin. NPY was measured using antibody RAS 7180. The limit of detection of the assay was 5 fmol/ml, and the IC50 was 110 ± 17 fmol/ml of original sample. The concentration of NPY in the samples ranged from 11 to 124 fmol/ml. The antibody does not cross-react with PYY, PP, NKA, SP, somatostatin, VIP, or [Met]enkephalin. For each sample, femtomoles per milliliter of neuropeptide measured by RIA were normalized to the wet weight of colonic tissue in each compartment and to the length of the collection period and expressed as femtomoles per 100 mg tissue wet weight per minute. Release during peristaltic reflex was calculated as the change in femtomoles per 100 mg per minute from release during a 15-min basal period. Student's t-test was used to assess stastically significant differences between control release and release in the presence of NPY and various receptor agonists and antagonists.
Materials. SP, VIP, NPY, the Y1 agonist [Leu31, Pro34]NPY, the Y2 agonist NPY13-36, the Y1-receptor antagonist BIBP 3226 {(R)-N2-(diphenylacetyl)-N-[(4-hydroxyphenyl)-methyl]-argininamide}, SP antibody RAS 7451, VIP antibody RAS 7161, NPY antibody RAS 7180, 125I-labeled VIP, 125I-labeled SP, and 125I-labeled NPY were purchased from Bachem-Peninsula (Torrance, CA). The Y2 antagonist BIIE 0246 {(S)-N2-[[1-[2-[4-[(R,S)-5,11-dihydro-6(6H)-oxodibenz[b,e]azepin-11-yl]-1piperazinyl]-2-oxoethyl]cyclopentyl] acetyl]-N-[2-[1,2-dihydro-3,5(4H)-dioxo-1,2-diphenyl-3H-1,2,4-triazol-4-yl]ethyl]-argininamide} was a gift from Boehringer Ingelheim Pharma (Biberach, Germany). All other chemicals and reagents were purchased from Sigma (St. Louis, MO).
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RESULTS
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Inhibition of NPY release during the peristaltic reflex. Basal release of NPY into the central and both peripheral compartments was similar, ranging from 1.18 ± 0.09 to 1.43 ± 0.17 fmol · 100 mg wet wt-1 · min-1. Stimulation of the mucosa in the central compartment at 4 and 8 strokes caused, respectively, 31.3 ± 6.2 (n = 4; P < 0.01) and 40.9 ± 5.9% (n = 4; P < 0.01) decrease in basal NPY release into the orad peripheral compartment where the ascending contraction was measured, but had no effect on NPY release into the central or peripheral caudad compartment (Fig. 1). Similarly, muscle stretch applied to the central compartment at 4 and 10 g caused, respectively, a 26.9 ± 4.4 and 40.0 ± 1.4% decrease in basal NPY release into the orad peripheral compartment only (Fig. 1).

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Fig. 1. Inhibition of neuropeptide Y (NPY) release during the peristaltic reflex. NPY release into the central (filled bar) and peripheral orad (open bar) and caudad (hatched bar) compartments of a 3-compartment flat-sheet preparation of rat colon was measured by RIA. The bathing medium was collected from each compartment over a 15-min control period and assayed for NPY (basal release). Mucosal stimuli (4 and 8 strokes) and muscle stretch (4 and 10 g) were then applied separately to the central compartment (5 times over a 15-min period), and the medium from all three compartments was collected and assayed for NPY. Significant inhibition of basal NPY was observed only in the orad compartment where ascending contraction was measured (open bars). Results are expressed as the change from basal level in fmol · 100 mg-1 · min-1 (range of basal levels: 1.2 ± 0.1 to 1.4 ± 0.2 fmol · 100 mg-1 · min-1). Values are means ± SE of 4 experiments.
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Selective inhibition of ascending contraction by NPY. As reported previously (15), mucosal stroking and muscle stretch applied to the central compartment elicited stimulus-dependent ascending contraction in the orad compartment and descending relaxation in the caudad compartment (Fig. 2). Addition of 1 µM NPY to the orad compartment inhibited ascending contraction elicited by mucosal stroking (60.7 ± 7.4% inhibition at 2 strokes to 33.1 ± 14.5% inhibition at 8 strokes) or muscle stretch (66.7 ± 16.7% inhibition at2gto 41.1 ± 4.8% inhibition at 10 g; Fig. 3). In contrast, addition of 1 µM NPY to the caudad compartment had no effect on descending relaxation elicited by mucosal stroking or muscle stretch (Table 1). Addition of NPY to the central compartment had no effect on ascending contraction or descending relaxation elicited by either form of stimulation (Table 2).

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Fig. 2. Tracing of orad and caudad responses of circular muscle and effect of Y1 and Y2 antagonist. Representative tracings of ascending contraction (top traces) and descending relaxation (bottom traces) of circular muscle recorded in the orad and caudad compartments, respectively. A stimulus of 4 strokes was applied to the mucosa of the segment in the central compartment. Ascending contraction was augmented by addition of the selective Y1 antagonist BIBP 3226 (1 µM) but not by the Y2 antagonist BIIE 0246 (1 µM).
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Fig. 3. Selective inhibition of ascending contraction by NPY. The peristaltic reflex was initiated by application of mucosal stimuli (2-8 strokes) or radial muscle stretch (2-10 g) to the central compartment of a 3-compartment flat-sheet preparation of rat colon. Ascending contraction and descending relaxation were measured in the orad and caudad peripheral compartments, respectively. NPY (final concentration 1 µM) was added variously to the central, orad, or caudad compartments. No effect of NPY on ascending contraction or descending relaxation was detected on addition to the central or caudad compartments for the entire range of stimuli (Tables 1 and 2). Selective inhibition of ascending contraction was observed on addition of NPY to the orad compartment. Results were expressed as %maximal response to mucosal stimulation (0.8 ± 0.1 g) or muscle stretch (0.9 ± 0.1 g). Values are means ± SE of 4 experiments for each modality of stimulation.
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Table 1. Lack of effect of NPY, and Y1 and Y2 agonists and antagonists on the peristaltic reflex when added to the caudad compartment
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Table 2. Lack of effect of NYP, and Y1 and Y2 agonists and antagonists on the peristaltic reflex when added to the central compartment
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Identification of the receptor type mediating the inhibitory effect of NPY on ascending contraction. Y1- and Y2-receptor agonists and antagonists were used to characterize the involvement of these receptors in the inhibitory action of NPY on ascending contraction. Addition of the preferential Y1-receptor agonist [Leu31, Pro34]NPY at a concentration of 1 µM to the orad compartment had a more striking inhibitory effect than NPY on ascending contraction induced by mucosal stroking or muscle stretch: inhibition ranged from 77.6 ± 14.7% at 2 strokes to 61.1 ± 3.2% at 8 strokes and from 77.8 ± 14.7% at 2 g to 71.0 ± 1.8% at 10 g (Fig. 4). Addition of the agonists to the orad compartment had no effect on descending relaxation (data not shown). Addition of the agonist to the caudad compartment (Table 1) or the central compartment (Table 2) had no effect on ascending contraction or descending relaxation elicited by either form of stimulation.

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Fig. 4. Selective inhibition of ascending contraction by Y1 receptor agonist. The peristaltic reflex was initiated by application of mucosal stimuli (2-8 strokes) or radial muscle stretch (2-10 g) to the central compartment of a 3-compartment flat-sheet preparation of rat colon. Ascending contraction and descending relaxation were measured in the orad and caudad peripheral compartments, respectively. The Y1-receptor agonist [Leu31, Pro34]NPY (final concentration 1 µM) was added variously to the central, orad, or caudad compartments. No effect of the Y1 agonist on ascending contraction or descending relaxation was detected on addition to the central or caudad compartments for the entire range of stimuli (Tables 1 and 2). Selective inhibition of ascending contraction was observed on addition of the Y1-receptor agonist to the orad compartment. Addition of the selective Y2-receptor agonist NPY13-36 (final concentration 1 µM) had no effect on ascending contraction or descending relaxation (Tables 1 and 2). Results were expressed as %maximal response to mucosal stimulation (1.0 ± 0.1 g) or muscle stretch (1.1 ± 0.1 g). Values are means ± SE of 7 experiments for each modality of stimulation.
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Conversely, addition of the selective Y1-receptor antagonist BIBP 3226 (7, 31) at a concentration of 1 µM to the orad compartment caused an increase in ascending contraction elicited by mucosal stroking or muscle stretch (Figs. 2 and 5) but had no effect on descending relaxation (data not shown). The increase in ascending contraction ranged from 93.3 ± 19.4% at two strokes to 41.9 ± 6.6% at eight strokes and from 54.3 ± 1.0% at 2 g to 13.7 ± 2.8% at 10 g. Addition of the antagonist to the caudad compartment (Table 1) or the central compartment (Table 2) had no effect on ascending contraction or descending relaxation elicited by either form of stimulation.

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Fig. 5. Selective augmentation of ascending contraction by Y1-receptor antagonist. The peristaltic reflex was initiated by application of mucosal stimuli (2-8 strokes) or radial muscle stretch (2-10 g) to the central compartment of a 3-compartment flat-sheet preparation of rat colon. Ascending contraction and descending relaxation were measured in the orad and caudad peripheral compartments, respectively. The Y1-receptor antagonist BIBP 3226 (final concentration 1 µM) was added variously to the central, orad, or caudad compartments. No effect of the Y1-receptor antagonist on ascending contraction or descending relaxation was detected on addition to the central or caudad compartments for the entire range of stimuli (Tables 1 and 2). Selective increase in ascending contraction was observed on addition of the Y1-receptor antagonist to the orad compartment. Addition of the selective Y2-receptor antagonist BIIE 0246 (final concentration 1 µM) had no effect on ascending contraction or descending relaxation. Results were expressed as %maximal response to mucosal stimulation (0.8 ± 0.1 g) or muscle stretch (1.1 ± 0.1 g). Values are means ± SE of 6 experiments for each modality of stimulation.
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In contrast, addition of the selective Y2-receptor agonist NPY13-36 or the selective Y2-receptor antagonist BIIE 0246 (6, 8) to the orad, caudad, or central compartment at a concentration of 1 µM had no effect on the ascending contraction and descending relaxation response elicited by mucosal stimulation or muscle stretch (Tables 1 and 2; Fig. 2).
Previous studies by us (13, 15) and others (5, 18, 22) have demonstrated that the ascending contraction component of the peristaltic reflex elicited by low levels of stimulation is mediated wholly by the release of acetylcholine from motor neurons whereas the response to more intense stimuli are mediated additionally by tachykinins. In the present study, we examined the effects of the Y1 agonist and antagonist in the presence of atropine to evaluate the role of the Y1 receptor in regulating the noncholinergic, tachykinin-mediated component of ascending contraction. Atropine (1 µM) abolished the response to low levels of stimulation (2 strokes and 2 g stretch) and inhibited the maximal response by 30 to 38%. The remaining ascending contraction response, mediated mainly by the tachykinin motor neurons, was affected by the Y1 agonists and antagonists in the same manner as described above. Addition of 1 µM of the Y1 agonist [Leu31, Pro34]NPY to the orad compartment inhibited ascending contraction elicited by mucosal stroking (abolition at 4 strokes to 55.6 ± 4.4% inhibition at 8 strokes) or muscle stretch (abolition at 4 g to 63.4 ± 5.8% inhibition at 10 g) (Fig. 6). In contrast, addition of 1 µM of the Y1 antagonist BIBP 3226 augmented the ascending contraction response elicited by mucosal stroking (108 ± 10% augmentation at 4 strokes to 28.7 ± 8.7% augmentation at 8 strokes) or muscle stretch (62 ± 8% augmentation at 4 g to 30.9 ± 5.7% augmentation at 10 g; Fig. 6).
Effect of Y1-receptor agonist and antagonist on excitatory (SP) and inhibitory (VIP) neurotransmitter release during the peristaltic reflex. Others (1, 5, 18, 22) and we (13, 15) have previously shown that ascending contraction of circular muscle is mediated by the combined effects of ACh and the tachykinins SP and NKA, whereas descending relaxation is mediated by the combined effects of VIP, PACAP, and NO (14, 15). In the present study, we measured SP release as a marker of excitatory neurotransmitter release during ascending contraction and VIP release as a marker of inhibitory neurotransmitter release during descending relaxation.
At a concentration of 1 µM, the Y1-receptor agonist [Leu31, Pro34]NPY had no effect on basal release of SP or VIP into any compartment. The agonist had no effect also on VIP release that accompanied descending relaxation elicited by mucosal stimulation or muscle stretch, but it inhibited SP release that accompanied ascending contraction elicited by mucosal stimulation or muscle stretch (Fig. 7). SP release during ascending contraction in the absence of the Y1-receptor agonist was 1.6 ± 0.4 and 3.0 ± 0.5 fmol · 100 mg-1 · min-1 above basal level at 4 and 8 strokes, respectively (basal SP release: 2.6 ± 0.2 fmol · 100 mg-1 · min-1). In the presence of the agonist, SP release elicited by 4 and 8 strokes was inhibited by 61 ± 7% (P < 0.01) and 42 ± 5% (P < 0.01), respectively (Fig. 7). Similarly, SP release in the absence of the agonist was 1.3 ± 0.1 and 1.9 ± 0.2 fmol · 100 mg-1 · min-1 above basal level at 4 and 10 g, respectively. In the presence of the Y1-receptor agonist, SP release elicited by 4 and 10 g was inhibited by 52 ± 5 (P < 0.01) and 46 ± 9% (P < 0.01), respectively (Fig. 7).

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Fig. 7. Selective inhibition of substance P (SP) release by Y1-receptor agonist. Mucosal stimuli (A; 4 and 8 strokes) or muscle stretch (B; 4 and 10 g) was applied to the central compartment of a 3-compartment flat-sheet preparation of rat colon. The Y1-receptor agonist [Leu31, Pro34]NPY (final concentration 1 µM) was added to the orad compartment, and the medium in this compartment was assayed for SP, whereas the medium in the caudad compartment was assayed for VIP. SP release elicited by mucosal stimuli or muscle stretch was significantly inhibited by the Y1-receptor agonist. Results are expressed as %basal release (basal SP: 2.6 ± 0.2 fmol·100 mg-1·min-1; basal VIP: 4.1 ± 0.2 fmol·100 mg-1·min-1). Control values depicted as open bars. Values are means ± SE of 4 experiments for each modality of stimulation. *P < 0.025 and **P < 0.01 from control.
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The effect of the Y1-receptor antagonist BIBP 3226 was the reverse of that of the agonist. At a concentration of 1 µM, the antagonist caused a small but significant increase (18.2 ± 4.3%; P < 0.025) in basal SP release but had no effect on basal VIP release. Consistent with its ability to augment ascending contraction, the antagonist increased SP release elicited by mucosal stimulation or muscle stretch in the orad compartment but had no effect on VIP release in the caudad compartment (Fig. 8). SP release elicited by four and eight strokes was increased by 54 ± 5 (P < 0.01) and 39 ± 9% (P < 0.025), respectively, whereas SP release elicited by 4 and 10 g was increased by 87 ± 13 (P < 0.01) and 42 ± 8% (P < 0.01), respectively (Fig. 8).

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Fig. 8. Selective augmentation of SP release by Y1-receptor antagonist. Mucosal stimuli (A; 4 and 8 strokes) or muscle stretch (B; 4 and 10 g) was applied to the central compartment of a 3-compartment flat-sheet preparation of rat colon. The Y1-receptor agonist BIBP 3226 (final concentration 1 µM) was added to the orad compartment, and the medium in this compartment was assayed for SP, whereas the medium in the caudad compartment was assayed for VIP. SP release elicited by mucosal stimuli or muscle stretch was significantly augmented by the Y1-receptor antagonist. Results are expressed as %basal release (basal SP: 2.3 ± 0.1 fmol · 100 mg-1 · min-1; basal VIP: 3.9 ± 0.2 fmol · 100 mg-1 · min-1). Control values depicted as open bars. Values are means ± SE of 4 experiments for each modality of stimulation. *P < 0.025 and **P < 0.01 from control.
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DISCUSSION
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This study provides the first evidence that NPY acts as a neurotransmitter in the intestine to regulate a physiological neuromuscular reflex. NPY appears to exert a tonic inhibitory influence of cholinergic/tachykinin neurons that mediate contraction of circular smooth muscle. This tonic inhibitory influence decreases during the ascending phase of the reflex and is reflected by a decrease in NPY release and an increase in SP release, a marker of other excitatory motor neurotransmitters, such as NKA, which is cosynthesized and coreleased with SP, and ACh which is colocalized and coreleased with both of these tachykinins (18). The inhibitory influence of NPY neurons is consistent with the established ability of NPY to inhibit ACh release in isolated nerve/muscle preparations. An earlier study by Holzer et al. (17) showed that exogenous application of NPY to an isolated segment of guinea pig small intestine inhibited ascending contraction. However, NPY release was not measured, and selective agonists or antagonists were not available then to permit analysis of the role of endogenous NPY in the regulation of the reflex.
In the present study, evidence for the functional role of NPY and identification of the Y receptor type mediating its effect were based on measurement of NPY release and on the use of selective Y1 and Y2 agonists and antagonists. The results indicated that NPY acted via Y1 receptors located on excitatory motor neurons. The conclusion is supported by immunochemical and Western blot analysis demonstrating expression of Y1 receptors in innervated muscle strip from the small intestine and colon of the rat (19, 24, 28) and by molecular analysis that disclosed the presence of mRNA encoding Y1 and Y4 receptors (11). The affinity of NPY or PYY for Y4 receptors, however, is very low, between 100- and 10,000-fold lower than for the natural ligand PP, which made it highly unlikely that Y4 receptors participated in the response to NPY (12, 25).
NPY and the selective Y1-receptor agonist [Leu31, Pro34]NPY inhibited, whereas the selective Y1-receptor antagonist BIBP 3226 augmented ascending contraction and SP release. Neither the Y2-receptor agonist NPY13-36 nor the antagonist BIIE 0246 had any effect on ascending contraction or SP release. NPY and the Y1- and Y2-receptor agonists and antagonists had no effect on descending relaxation or VIP release. The results imply that NPY, acting via Y1 receptors, inhibits the release of excitatory motor neurotransmitters mediating ascending contraction of circular muscle. The results further imply that in the basal state, NPY exerts a tonic inhibitory restraint on excitatory motor neurons that is relieved during the ascending phase of the reflex, and that is reflected by a decrease in NPY release and an increase in the release of excitatory neurotransmitters. The pathway that leads to a decrease in the activity of NPY neurons has not been identified. One possibility is that NPY neurons are regulated by cholinergic interneurons, which control their activity and provide additional direct excitatory input to motor neurons.
There is a noteworthy parallelism between the role of NPY in regulating the ascending pathway and the role of [Met]enkephalin in regulating the descending pathways. In earlier studies (16), we showed that opioid neurons exert a tonic restraint on inhibitory VIP/PACAP/NOS motor neurons. This restraint is relieved during the descending phase via an increase in the activity of somatostatin interneurons that synapse with these opioid neurons. Consequently, opioid agonists inhibited and opioid antagonists augmented VIP and NO release and descending relaxation.
In previous studies, others (1, 5, 18, 22) and we (13, 15) have shown that the ascending contraction is mediated by release of ACh and the tachykinins SP and NKA. ACh is released by mucosal stimulation and muscle stretch of both low and high intensity, whereas SP and NKA are coreleased by high-intensity stimulation (13). Consequently, atropine inhibited the contractile response to low- and high-intensity stimuli, whereas tachykinin antagonists and antibodies to SP and NKA inhibited only the response to high-intensity stimuli. In the present study, the inhibitory effects of NPY and Y1-receptor agonists and the stimulatory effects of Y1-receptor antagonists were observed at all levels of stimulation, implying that NPY regulates the release of ACh and the tachykinins. Inhibition of ACh release from activated cholinergic neurons in nervemuscle preparations of the intestine and colon is a well-established pharmacological property of NPY. In the present study, atropine abolished the response to the lowest levels of mucosal stimulation and to muscle stretch, confirming that they are wholly mediated by the release of ACh. The ability of NPY and Y1 agonists to inhibit and of Y1 antagonist to augment these responses to low levels of stimulation indicates that the Y1 receptor does regulate ACh release during the ascending phase of the peristaltic reflex. The ascending contraction that remained in the presence of atropine (i.e., the noncholinergic component of ascending contraction mediated by tachykinin motoneurons) was also inhibited by the Y1 agonist and augmented by the Y1 antagonist. These findings were directly confirmed by measurement of SP release. The ability of the Y1 agonist and antagonists to produce the same effect in the presence and absence of atropine indicates that the Y1 receptors are present on motor neurons that release ACh and on those that release both ACh and tachykinins. The ability of the Y1 agonist and antagonists to produce the same effect in the presence of atropine also indicates that the regulation of SP release is mediated directly rather than indirectly through an effect on ACh release. It is important to emphasize that the physiological action of NPY depends on its access to specific pre- or postjunctional receptors located at the site of release. The present study did not identify the site of action of NPY as pre- or postjunctional, although a recent immunohistochemical study (24) suggests that Y1 receptors are located in nerve cell bodies in the myenteric plexus as well as on nerve fibers innervating the circular muscle layer of rat small intestine. Either location is consistent with the physiological actions of NPY identified in the present study.
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DISCLOSURES
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-34153.
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FOOTNOTES
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Address for reprint requests and other correspondence: J. R. Grider, Dept. of Physiology, Medical College of VA Campus, Virginia Commonweath University, P.O. Box 980551, Richmond, VA 23298 (E-mail: jgrider{at}hsc.vcu.edu).
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. Section 1734 solely to indicate this fact.
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