Interaction of xenin with the neurotensin receptor of guinea pig enteral smooth muscles

Peptides 23 (2002) 1519–1525

Interaction of xenin with the neurotensin receptor of guinea pig enteral smooth muscles夽 Gerhard E. Feurle∗ , Jörg W. Metzger, Alexandra Grudinki, Gerd Hamscher DRK-Krankenhous Neuwied, Medizinische Klinik, Rheinische Friedrich-Wilhelms Universität, Bonn, Germany Received 15 August 2001; accepted 19 October 2001

Abstract Xenin, a 25 amino acid peptide, interacts with the neurotensin receptor subtype 1 of intestinal muscles of the guinea pig. Replacement of the C-terminal Lys–Arg peptide bond in xenin 6 by a reduced pseudo-peptide bond augmented binding affinity to isolated jejunal and colonic muscle membranes by factors of 7.7 and 21.0 respectively; the potency to contract the jejunum and to relax the colon was increased by factors of 3.2 and 1.3. The C-terminus Trp-Ile-Leu (WIL) of xenin, in contrast to the C-terminus Tyr-Ile-Leu (YIL) of neurotensin, bound competitively to the muscle membranes. WIL blocked the contractile action of xenin in the jejunum and was synergistic with the relaxing action in the colon. The Lys–Arg motif and Trp in the C-terminus of xenin are essential structures in the action of xenin on the enteral smooth muscle receptors. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Xenin; Neurotensin; Neurotensin receptor; Smooth muscle; Guinea pig; Intestine; Reduced ␺ pseudo-peptide bond

1. Introduction Three receptor subtypes have been cloned for the peptide neurotensin [5,22,23,29]. Neurotensin receptor subtype1 (NTS1 ) is characterized by the non-peptide antagonist SR 48692 [16,27], neurotensin receptor subtype2 (NTS2 ) by the histamine 1 receptor antagonist levocabastine [5,22], and the most recently cloned neurotensin receptor subtype3 (NTS3 ) is homologous with previously identified human gp 95/sortilin [23]. The mammalian peptide ligands to these receptor subtypes are neurotensin and neuromedin N [30]. Neurotensin is a 13 amino acid regulatory peptide originating in various regions of the central nervous system [3] and in the N cells of the ileal mucosa [4,18]. Neuromedin N is a structurally related analogue to neurotensin isolated from the spinal cord of pigs [24]. Rarely mentioned is another mammalian peptide ligand, the 25 amino acid peptide xenin [6,11,12,17]. Xenin is produced by specific endocrine cells of the duodenal mucosa [1]. It circulates in the human plasma [11,15] and is active in stimulating exocrine pancreatic secretion, in inhibiting pentagastrin-induced gastric 夽

PII of original article S0196-9781(01)00637-4. Corresponding author. Present address: DRK-Krankenhaus Neuwied, Markstr. 74, 565654 Neuwied, Germany. Tel.: +49-2631-981401; fax: +49-2631-981490. E-mail address: [email protected] (G.E. Feurle). ∗

secretion of acid, and in stimulating intestinal motility in the dog [13]. In humans, i.v. infusion of xenin induces phase III-like contractions of the interdigestive motor complex of the jejunum [15]. In the guinea pig, xenin elicits in vitro a neurokinetic excitatory effect on jejunal muscle strips with participation of muscarinic, purinergic, and tachykinin-related mechanisms. In the colon, it elicits a myokinetic relaxing effect involving Ca2+ -dependent K+ channels and the P2 purinoceptor [12]. The rat ileum is relaxed by xenin, mediated by an apamin-sensitive mechanism [6]. The effects of xenin on small and large bowel are inhibited by the non-peptide neurotensin receptor antagonist SR 48692 [6,12]. This indicates that xenin exerts its contractile and relaxing effects via the NTS1 . NTS1 has so far be cloned only in the central nervous system [29]. The presence of the mRNA of NTS1 has, however, been shown by in situ hybridisation also in the intestine of rats and humans [29,31]. Our data that SR 48692 inhibits the effects of xenin in the guinea pig bowel indicate that NTS1 is present and functionally active in the intestinal muscle of this species. To our knowledge, no data are available as to the presence of NTS2 and NTS3 in the intestines. The interaction of xenin with the neurotensin receptor did not come as a surprise since the amino acid sequences of the biologically active C-termini of xenin and neurotensin are structurally related: they share a free non-amidated C-terminal isoleucine–leucine sequence and a pair of basic

0196-9781/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 1 9 6 - 9 7 8 1 ( 0 2 ) 0 0 0 6 4 - 5

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2.2. Receptor binding study

Fig. 1. Amino acid sequence of the peptides and pseudo-peptides investigated.

amino acids flanked by two prolines. Substitution of Arg by Lys as one of the basic amino acids and of Tyr by Trp are the only differences in the C-terminus of xenin, whereas the mid-sequences and the N-termini of xenin and neurotensin show no homologies (Fig. 1). Previous studies have suggested that the pair of arginines stabilised by two adjacent prolines in neurotensin is essential for ionic interaction with the neurotensin receptor [10]. The biological activities of xenin and of neurotensin are very similar [14,19]. In vitro studies, however, have also revealed differences: the dose–response curves in the contraction of the guinea pig jejunum were not parallel; neurotensin has a reduced potency and an increased efficacy as compared to xenin. In the relaxation of the precontracted colon, neurotensin was significantly more potent than xenin [12]. It is the aim of the present investigation to study in greater detail the interaction of the biologically active C-terminus of xenin with the muscular receptors of the small and large intestine of the guinea pig. Our interest was focused on the peptide bond of the dibasic Lys–Arg motif and on the role of the non-polar hydrophobic amino acid Trp in xenin in lieu of the uncharged polar amino acid Tyr in neurotensin.

The assay was performed as described by Labbé-Jullié et al. [20]. Segments of jejunum and colon were obtained from 2 to 3 months old female inbred guinea pigs immediately after a blow to the neck and ex vivo removal of the heart for cardiac research purposes. The gut segments were opened, cleaned, scraped free of mucosa, and washed three times. The remaining muscle tissue was homogenised using an Ultraturrax in 10 volumes of 5 mM Tris–HCl, pH 7.5 containing 5 mM EDTA and centrifuged at 125,000 × g for 30 min. The pellet was washed twice and the final pellet was suspended in 2 ml of 5 mM Tris–HCl pH 7.5 per gram of tissue and kept frozen at −18 ◦ C. It could be used for several months. For the binding assay, 50 ␮l suspended homogenate was incubated with 200 ␮l buffer (50 mM Tris–HCl, 1 mM MgCl2 ·6H2 O, 0.8 mM phenanthrolen, 0.2% bovine serum albumin (BSA)). This suspension was incubated at room temperature with 0.08 nM 125 I-xenin analogue (100,000 cpm) with a specific activity of 2000 Ci/mM for 30 min. Receptor binding was terminated by addition of 2 ml of ice cold incubation buffer. The incubate was then manually pumped through 0.2 ␮m cellulose acetate filters (Sartorius). The filters were washed twice with 2 ml incubation buffer. Non-specific binding was determined after addition of 1 ␮M unlabelled xenin to the incubate. Increasing concentrations of xenin 25, xenin 6,
xenin 6, WIL, IL, LIW, and of the neurotensin receptor antagonist SR 48692 were added to the incubation medium in individual experiments. The equilibrium constant was calculated as suggested [8]. The binding experiments at each ligand concentration were performed six-fold. The results were expressed as percentage of initial specific binding. EC50 and IC50 were defined as the concentration needed to obtained 50% of the maximal response that could be obtained with the substance tested.

2. Material and methods

2.3. Contractility study

2.1. Substances

The technique to investigate isolated guinea pig muscle strips of the jejunum and colon has been reported before [12]. In short, segments of 2–3 cm length of isolated jejunum and colon were longitudinally suspended under defined resting tension in oxygenated Krebs–Henseleit buffer. The colonic strips were precontracted with 1 ␮M metacholine. The changes in length of the strips were recorded immediately after addition of the active agent to the incubation medium. The strips were washed as soon as a maximum effect had occurred. A new stimulus was injected 10 min after a second wash, when the strip length had returned to baseline. When the interaction of two agents was studied, i.e. xenin and WIL, the agents were injected 20 s apart from each other without washing. Changes in length were recorded by an isotonic transducer; the results were normalised on the basis of the response to 1 ␮M metacholine given as 100%.

Xenin 25 was custom-synthesised by Bachem Inc. (Los Angeles CA) according to the sequence reported [11]. The fragments xenin 23–25 Trp-Ile-Leu (WIL) the reverse sequence Leu-Ile-Trp (LIW) and the C-terminal tripeptide of neurotensin Tyr-Ile-Leu (YIL) were synthesised as described [12]. The fragments 20–25 of xenin (xenin 6) and a non-peptide analogue in which the Lys–Arg peptide bond was replaced by a
-CH2 –NH reduced bond (
xenin 6) (pronounced: psi) were synthesised by Saxon Biochemicals Hannover, Germany. The radiolabelled xenin analogue was prepared as reported earlier [11]. The neurotensin receptor antagonist SR 48692 was donated by Sanofi (Toulouse, France). Levocabastine was donated by Janssen Pharma, Neuss. The sequences of the various peptides and pseudo-peptides are shown in Fig. 1.

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Animal tissue was obtained in accordance with the German Federal Law for Animal Welfare (Tierschutzgesetz). 2.4. Statistics Student’s test for paired values was used for statistics. P < 0.05 was set as significant.

3. Results 3.1. Binding study Increasing concentrations of xenin 25 competitively reduced the binding of radiolabelled xenin 25 analogue to homogenised muscle tissue of the guinea pig jejunum and colon (Fig. 2). Non-specific binding was less than 1%. The specific neurotensin receptor antagonist SR 48692 led to a shift to the right of the concentration–response curve. Doses of approximately 10−6 M of xenin 25 and of SR 48692 reduced the binding of the tracer to almost zero (Fig. 2). Xenin 6 and
xenin 6 elicited parallel displacement curves;
xenin 6, however, was 7.7 times more potent in binding to the isolated jejunal muscle membranes and 21 times more potent in binding to colonic muscle membranes than the corresponding xenin 6 fragment (Fig. 3, Table 1). The C-terminal tripeptide of xenin WIL reduced the binding of radiolabelled xenin analogue to homogenised muscle tissue of the guinea pig jejunum and colon with an equilibrium constant (Ki ) of 1590 and 252 nM, respectively, whereas the reversed tripeptide LIW and the C-terminal tripeptide of neurotensin YIL had no effect (Ki > 10,000) (Fig. 4, Table 2). The equilibration constants (nM) expressing the concentration producing half maximum inhibition of specific binding of 125 I-xenin analogue to the receptor at the guinea pig bowel muscles are shown in Table 2. Levocabastine in doses

Fig. 2. Competitive inhibition of 125 I-xenin analogue-specific binding to homogenates of guinea pig jejunum (A) and colon (B) by xenin 25 and the neurotensin receptor antagonist SR 48692.

Fig. 3. Competitive inhibition of 125 I-xenin analogue-specific binding to homogenates of guinea pig jejunum (A) and colon (B) by xenin 25, the hexapeptide fragments
xenin 6 and xenin 6.

up to 10−6 M did not reduce the binding of the radiolabelled xenin 25 analogue (Table 2). 3.2. Contractility study Xenin contracted the isolated guinea pig jejunum dose dependently and relaxed the precontracted guinea pig colon. Both effects were antagonised by the non-peptide receptor antagonist SR 48692 to neurotensin (Fig. 5). Xenin 6 induced concentration–response curves that were shifted to the right parallel to xenin 25. The dose–response curve induced by
xenin 6, however, was significantly steeper than that of xenin 6 and xenin 25 (Fig. 6). The efficacy of
xenin 6 in contracting the jejunum was significantly greater than that of xenin 6 and xenin 25. The potency of
xenin 6 was 3.2 times greater in contracting the jejunum (P < 0.001) and insignificantly more potent in relaxing the colon than xenin 6 (P < 0.061) (Table 1).

Fig. 4. Competitive inhibition of 125 I-xenin analogue-specific binding to homogenates of guinea pig jejunum (A) and colon (B) by xenin 25, the tripeptides Trp-Ile-Leu (WIL) of xenin. The reverse tripeptide Leu-Ile-Trp (LIW) and the tripeptide Tyr-Ile-Leu (YIL) of neurotensin do not show a competitive displacement of the tracer.

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Table 1 Equilibrium and potency parameters of the C-terminal hexapeptide sequence of xenin (xenin 6) and of the reduced pseudo-peptide
xenin 6 analogue in binding to isolated guinea pig muscle membranes and in contracting or relaxing isolated strips of guinea pig small and large intestine Xenin 6


Xenin 6

Augmentation factor

P

Binding study equilibrium constant (nM) Ki (M ± S.E.M.) Jejunum (n = 7) Colon (n = 5)

4.34 ± 0.98 1.68 ± 0.73

0.56 ± 0.21 0.08 ± 0.02

7.7 21.0

<0.001 <0.001

Contraction/relaxation potency (nM) EC50 (M ± S.E.M.) Jejunum (n = 5) Colon (n = 5)

14.9 ± 1.46 8.48 ± 1.52

4.64 ± 0.82 6.58 ± 1.22

3.2 1.3

<0.001 0.061, n.s.

80 ± 8 85 ± 7

115 ± 11 96.0 ± 7

1.4 1.1

Efficacy % (M ± S.E.M.)

Jejunum (n = 5) Colon (n = 5)

0.001 0.029

n.s.: not significant.

Table 2 Equilibrium and potency parameters of neurotensin, xenin 25, the neurotensin receptor 1 antagonist SR 48692, the C-terminal tripeptide WIL of xenin, the C-terminal tripeptide YIL of neurotensin, the reverse tripeptide LIW and the neurotensin receptor 2 antagonist levocabastine in binding to isolated guinea pig muscle membranes and in contracting or relaxing isolated strips of guinea pig small and large intestine Neurotensin

Xenin 25

SR 48692

WIL

YIL

LIW

Levocabastin

Binding study equilibrium constant Ki (nM) Jejunum 0.30 ± 0.07 Colon 0.13 ± 0.03*

0.37 ± 0.09 0.17 ± 0.09

6.89 ± 2.34 3.07 ± 0.62

1590 ± 1100 252 ± 80

>10 000 >10 000

>10 000 >10 000

>10 000 >10 000

Contraction/relaxation potency EC50 (nM) Jejunum Colon

2.04 ± 0.28 3.14 ± 0.52

25.6 ± 11.0 38.5 ± 15.3

n.e. n.e.

n.t. n.t.

n.t. n.t.

n.t. n.t.

5.19 ± 2.49 1.19 ± 0.98

M ± S.E.M., n.e. = no effect measurable, n.t. = not tested. *From Labb´e-Julli´e et al. [20].

The contraction of the guinea pig jejunum induced by 10−7 M xenin 25 was abolished when WIL in concentrations of 10−7 , 10−6 , and 10−7 M was injected into the incubation bath 20 s earlier (Fig. 7). Responsiveness of the muscle strips to xenin 25 was restored after washing. Injection of two doses of 10−7 M xenin 25 into the incubation medium 20 s apart from each other induced additive effects. A sec-

Fig. 5. Dose–response curves of xenin 25, neurotensin and the combination of xenin 25 + the neurotensin receptor antagonist SR 48692 on contraction of the isolated guinea pig jejunum (A) and on relaxation of the precontracted isolated guinea pig colon (B). This figure is reproduced in part from a previous publication [12] with permission of the Publisher.

ond wash restored the responsiveness to 10−7 M xenin. This sequence was tested in nine experiments. Three of these are shown in Fig. 7. In the precontracted colon of guinea pig, WIL, in a dose of 10−5 M and injected 20 s prior to 10−7 M xenin 25, showed an additive relaxing effect. An example is shown in Fig. 7.

Fig. 6. Dose–response curves of the effect of xenin 25 and the hexapeptide
xenin 6 and the hexapeptide xenin 6 on contraction of the isolated guinea pig jejunum (A) and on relaxation of the precontracted isolated guinea pig colon (B).

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Fig. 7. Contraction of three isolated strips of jejunum (A–C). Xenin 25 in a concentration of 10−7 M (X−7 ) induces a contraction in all segments (1st row, upward reflection). WIL in concentrations of 10−7 M (x−7 ), 10−6 M (x−6 ), and 10−5 M (x−5 ) induces no visible contraction but inhibits the effect of 10−7 M xenin 25 (X−7 ) injected 20 s later (2nd row). After washing, the effect of 10−7 M xenin 25 (X−7 ) is restored (3rd row). The effect of 10−9 M xenin 25 (X−9 ) induces a submaximal contraction that is augmented by an injection of 10−7 M xenin 25 (X−7 ) 20 s later (4th row). After washing, the effect of 10−7 M xenin 25 (X−7 ) is restored (5th row). (D) A colonic strip of the guinea pig is contracted by 10−6 M metacholine (M−6 ) (upward reflection). An injection of 10−5 M tripeptide xenin 3 (WIL) (x−5 ) induces a relaxing effect (downward reflection) that is augmented by 10−7 M xenin 25 (X−7 ) injected 20 s later.

4. Discussion The insertion of a reduced pseudo-peptide bond into an amino acid sequence alters its conformation and can thereby change the affinity to a receptor. It may, however, also increase biological activity by raising resistance to enzymatic degradation [7]. The strikingly enhanced potency of the C-terminus of xenin to bind to intestinal muscle membranes and to contract and relax the small or large bowel after insertion of a pseudo-peptide bond between the two basic amino acids Lys and Arg indicates a crucial role of this dibasic motif in the interaction and/or degradation of xenin at the enteral neurotensin receptor. A similar phenomenon has previously been described for neurotensin [21]. The reduced H-Lys
-(CH2 –NH) Lys-Pro-Tyr-Ile-Leu OH analogue of neurotensin was ten times more potent than the fragment neurotensin 8–13 in contracting the isolated guinea pig ileum. Binding to newborn mouse brain homogenates, however, was not changed. The increased potency of the
8,9-neurotensin compound correlated to its resistance to degradation. It is of interest that introduction of the
pseudo-bond conferred increased potency only when it took place within the pair of basic amino acids. Reduced pseudo-peptide bonds at other locations within the 8–13 neurotensin fragment decreased potency. The insertion of the reduced
bond in the dibasic motif of xenin increases the binding affinity to isolated mus-

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cle membranes of the jejunum and colon to a greater extent than the biological potency to contract and relax the bowel. Proteases are expected to be more active when intact bowel segments are incubated at 37 ◦ C, without precautions to inhibit enzymes than when homogenised and centrifuged muscle membranes are incubated in the presence of the metallopeptidase inhibitor phenantrolene at 20 ◦ C. Our observation suggests, therefore, that the Lys–Arg motif of xenin is perhaps of greater importance in the alignment of xenin to its receptor than in the protection of xenin against enzymatic degradation. The dibasic motif is also required for prohormone sorting to the regulated secretory pathway [9]. The reduced
Lys–Arg bond in xenin increased the potency of this hexapeptide analogue to contract the jejunum and relax the colon not only as compared to the hexapeptide xenin 6 but
xenin 6 was almost as potent as the parent molecule xenin 25. Moreover the slope of the dose–response curves was significantly steeper than that of xenin 25 and of xenin 6, and the efficacy of the reduced xenin 6 analogue in contracting the jejunum was even greater than that of xenin 25. These observations underscore the unique role of the Lys–Arg bond in the interaction and degradation of xenin with its receptor. It should be stressed, however, that the enteral NTS1 in the guinea pig with the two subtypes (neurokinetic in the small and myokinetic variant in the large bowel [12] may differ from the NTS1 cloned from cerebral tissue. The almost selectively increased potency and efficacy of
xenin 6 to contract the guinea pig jejunum and our previous findings that xenin 25 initiates strong propagating phase III-like contractions of the human jejunum [15], indicate a potential role for this or a related pseudo-peptide analogue in the treatment of bowel paralysis in humans. Pseudo-peptides with reduced peptide bonds arc promising therapeutic agents in the treatment of various other disorders [2,25,28]. The replacement of tyrosine in the C-terminus of neurotensin by tryptophane in xenin induces an alteration in the peptide/receptor interaction that is most evident when the isolated tripeptides are examined. The C-terminal tripeptide of xenin WIL, in contrast to the C-terminal tripeptide YIL of neurotensin binds to muscle membranes of the guinea pig jejunum and colon and is bioactive when tested in isolated bowel segments. In the contractility study, WIL in dilutions up to 10−7 M does not induce contractions of the guinea pig jejunum [12]. However, the sequence WIL unexpectedly exerts inhibitory biological activity when applied immediately before an excitatory dose of xenin 25. In the colon, where the action of xenin on contractility is reverse from its action in the jejunum, a high dose of the tripeptide WIL also induced a biological action by inducing an additive relaxing effect (Fig. 7). Levocabastine did not affect the binding of radiolabelled xenin analogue, indicating that only the high affinity binding receptor NTS1 was engaged in the binding reactions. This conclusion is supported by the low equilibrium constant of xenin 25 of 0.17 nM.

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It appears that the tripeptide WIL holds xenin at its jejunal receptor binding site without exerting contractile effects and thereby blocks the action of additional xenin 25. Molecular modelling of neurotensin suggests that the hydroxy group of Tyr11 interacts with the two guanidinium groups of Arg8 and Arg9 and thus forms a specific three-dimensional structure that binds to seven aromatic residues in the third extracellular loop of human and rat NTS [26]. As in xenin, Tyr is replaced by Trp, the three-dimensional structure of xenin will be unlike that of neurotensin and the interaction of neurotensin and xenin with the receptor may be different. Our findings lend support to the concept that, although xenin and neurotensin act on an identical enteral (NTS1 ) receptor [6], the ligand–receptor interaction is not identical [12]. One determinant of this difference seems to be the replacement of Tyr in neurotensin by Trp in xenin in the biologically important C-terminal sequence of these peptides. Apart from this difference in binding and biological action of the C-termini of neurotensin and xenin, the tripeptide WIL is, in vitro, a bioactive molecule.

Acknowledgments This work was supported by the Deutsche Forschungsgemeinschaft (Fe 127/9). We are indebted to J.A.J. Schuurkes, Dept. of Gastrointestinal Pharmacology, Janssen Research Foundation Beerse, Belgium and T. Sauerbruch, Medizinische Kimik University of Bonn, Germany, for their generous support and permission to perform this investigation in their laboratories. Our work was not supported by the PSI mystery series. Parts of Fig. 5 are reproduced from a previous publication in the J. Pharm. Exp. Ther. [12] with permission of the Publisher. The present address of Dr. Hamscher is Tierarztliche Hochschule Hannover, Abt. Lebensmitteltoxikologie.

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