European Journal of Pharmacology, 175 (1990) 43-47

43

Elsevier EJP 51126

Comparison of neuromedin-N and neurotensin on net fluid flux across rat small intestine R a c h e l A. Spokes

1 Ying

C. Lee, Y i a n g o s Y i a n g o u , J a n D o m i n a n d S t e p h e n R. B l o o m

Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W12 ONN, U.K.

Received 17 October 1989, accepted 24 October 1989

Neuromedin-N, a hexapeptide recently isolated and purified from porcine spinal cord, has close sequence homology with the C-terminal region of the tridecapeptide neurotensin. Both peptides have a remarkably similar peripheral distribution. Little is known of the biological activity of neuromedin-N. Neurotensin and peptide histidine methionine are known to stimulate net fluid secretion into rat small intestine. We have therefore tested the effect of neuromedin-N and the hexapeptide neurotensin-(8-13), the smallest fully active analogue of neurotensin in this system, compared with that of neurotensin and peptide histidine methionine. All four peptides reduced net absorption in low doses and caused net secretion in larger doses. However, whereas peptide histidine methionine was active in all areas of the small intestine, neurotensin, neurotensin-(8-13) and neuromedin-N were inactive in the duodenum. In the post-duodenal areas neurotensin was approximately 7 times more active than peptide histidine methionine, 21 times more potent than neuromedin-N and 33 times more potent than neurotensin-(8-13). Neurotensin; Neuromedin-N; Water absorption; Small intestine

1. Introduction Neuromedin-N (Lys-Ile-Pro-Tyr-Ile-Leu) is a hexapeptide, isolated and purified from porcine spinal cord (Minamino et al., 1984). It has close sequence homology with the six C-terminal amino acids of neurotensin (pGlu-Leu-Tyr-Gln-Asn-LysPro-Arg-Arg-Pro-Tyr-Ile-Leu), a tridecapeptide widely distributed in the mammalian central nervous system and gastrointestinal tract (Carraway and Leeman, 1976; Holzer et al., 1982). The peripheral distribution of neuromedin-N has recently been shown to be remarkably similar to

1 Present address: Wyeth Laboratories, Huntercombe Lane South, Taplow, Maidenhead, Berks. SL6 0PH, U.K. Correspondence to" S.R. Bloom, Department of Medicine, 2nd Floor, Francis Fraser Laboratory, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W12 0NN, U.K.

that of neurotensin (Carraway and Mitra 1987; Lee et al., 1987) and both peptides were subsequently shown to be processed from the same precursor (Dobner et al., 1987). Neuromedin-N enhances the contractility of guinea-pig ileum with about 15% of the activity of neurotensin (Minamino et al., 1984) and has recently been shown to increase pancreatic exocrine secretion and pancreatic, mesenteric and portal blood flow (Sumi et al., 1987). Neuromedin-N also binds to brain neurotensin receptors with the same affinity but a 19-fold lower potency than neurotensin (Checler et al., 1986), Neurotensin is a potent stimulant of net fluid secretion into the lumen of the small intestine of the anaesthetised rat (Mitchenere et al., 1981) but its relative activity in different areas of the small intestine and the effect of neurotensin on fluid secretion are not known. We have therefore studied the activity of neurotensin and neuromedin-N in

0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

44 five areas of the small intestine of the anaesthetised rat. Only the first two amino acids of neuromedin-N differ from those of neurotensin. It was, therefore, decided to test neurotensin-(8-13) (i.e. the C-terminal 6 amino acids of neurotensin) which is the same length as neuromedin-N in order to determine whether the length or the amino acid sequence of neurotensin is most important for its biological activity. In addition the effects of these three peptides have been compared with those of peptide histidine methionine which has previously been shown to be active in this model (Spokes et al., 1988). The possibility that low doses of neurotensin and peptide histidine methionine might potentiate each other has also been investigated.

2. Materials and methods

The method was adapted from one used previously (Ghiglione et al., 1982). Female Wistar rats (180-240 g) were starved of solid food for 18-24 h but given free access to water. They were anaesthetised with 2.7 m l / k g of fentanyl citrate (0.08 m g / m l ) , pre-mixed with fluanisone (2.5 m g / m l ) (Hypnorm, Janssen Pharmaceuticals Ltd., Oxford, U K ) and midazolam (1.25 m g / m l ) (Hypnovel, Roche Products Ltd., Welwyn Garden City, UK), i.p. A heat pad was used to maintain the temperature of the rat at 37-38°C. The left femoral vein was cannulated and the animal infused with saline (154 nM NaC1) at 2 m l / h . The abdomen was opened through a midline incision and segments of the small intestine, 5 cm in length, were isolated with a cotton ligature at each end. One in the duodenum, three evenly spaced along the jejunum and one in the ileum. Each segment was injected with 0.5 ml of Krebs solution, composition (nM): NaC1 118, KC1 4.7, CaC12 2.5, MgSO 4 1.2, K H 2 P O 4 1.2, N a H C O 3 2.5 and glucose 11. The abdomen was closed and infusion of peptide or control solution started. Synthetic peptide histidine methionine, neurotensin, neurotensin-(8-13) or neuromedin-N (Peninsula Laboratories Inc., St Helens, Merseyside, U K ) were dissolved in saline solution containing 1% bovine serum albumin and infused for 40 min at 2 m l / h using a syringe ram p u m p (Harvard Apparatus, Millis, MA, USA).

Three doses of each peptide were tested which were chosen, by prior pilot experiment, to give approximately equal responses for each peptide. Six to ten rats received each dose. Eight control rats received vehicle alone. At the end of the 40 min infusion the rats were killed, the isolated gut loops removed and weighed before and after being cut open, drained and gently blotted. Fluid accumulation or loss in each segment was calculated as m l / g of blotted tissue weight, assuming 1 ml of fluid to weigh 1 g. Net fluid transport was expressed as mean change (_+ S.E.M.) of luminal fluid in m l / g per 40 min for each group of segments in equivalent positions. For overall peptide comparisons the results for the four post-duodenal segments of each rat were meaned to give a single value. Dose-response curves were then constructed for the three peptides. Dose ratios for the activity of the peptides were calculated using one-way analysis of variance.

3. Results

Fluid absorption in the control rats decreased along the gut. In the jejunum and ileum low doses of neurotensin, neuromedin-N or neurotensin-(8113) caused a reduction in net absorption while larger doses caused net secretion of fluid into the gut neuromedin-N, neurotensin and neurotensin(8-13) were inactive in the duodenum. Peptide histidine methionine, however, was active in all areas of the small intestine. Figure 1 shows the effect of three doses of each peptide at the five sites along the gut. Figure 2 shows the results for the post-duodenal segments, portrayed as dose-response curves. Oneway analysis of variance of these results indicated that neurotensin was 21.1 times more potent than neuromedin-N with 95% confidence limits of 14.9 and 30.6 (n = 6-10), 33.1 times as potent as neurotensin-(8-13) (95% confidence limits 22.2-58.0) and 7.2 times more active than peptide histidine methionine (95% confidence limits 5.0-11.1). Neuromedin-N was 1.6 times more potent than neurotensin-(8-13) (95% confidence limits 1.2-12.5), (P < 0.05).

45

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ILEUM

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~

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Peptide Histidine Methionine

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Fig. 1. The effects of three doses each of neurotensin, n e u r o m e d i n - N , neurotensin-(8-13) a n d p e p t i d e h i s t i d i n e m e t h i o n i n e on fluid a b s o r p t i o n ( - ve) a n d secretion ( + ve) in five areas of the small i n t e s t i n e of the a n a e s t h e t i s e d rat. Results are m e a n s + S.E.M. from six to ten rats. * P < 0.05; * * P < 0.01 c o m p a r e d w i t h control (two-tailed s t u d e n t ' s t-test).

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Nominal Infusion Rate (pmol/kg/min)

Fig. 2. Dose-response curves for the effects of neurotensin, p e p t i d e histidine m e t h i o n i n e , n e u r o m e d i n - N a n d neurotensin(8-113) on net fluid a b s o r p t i o n ( - v e ) a n d secretion ( + ve) in the small intestine of the a n a e s t h e t i s e d rat. Results p l o t t e d are the m e a n value for the four p o s t - d u o d e n a l segments of each rat ( + S.E.M.) against the n o m i n a l infusion rate.

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Fig. 3. The effect of p e p t i d e h i s t i d i n e m e t h i o n e (100 p m o l / k g per min), n e u r o t e n s i n (20 p m o l / k g per min) and p e p t i d e histidine m e t h i o n i n e + n e u r o t e n s i n on net fluid m o v e m e n t across the small i n t e s t i n e of the a n a e s t h e t i s e d rat c o m p a r e d w i t h control ( - ve values i n d i c a t e a b s o r p t i o n a n d + ve values secretion). R e s u l t s are m e a n s _+S.E.M. from six to ten rats. * P < 0.05; * * P < 0.01 c o m p a r e d w i t h control (two-tailed S t u d e n t ' s t-test).

46 Figure 3 compares the effect of low, equi-active doses of peptide histidine methionine and neurotensin, given alone and together, compared with control at the same five sites along the gut. When the responses to the four post-duodenal segments are meaned (as for fig. 2), the mean response to the 100 p m o l / k g per min of peptide histidine methionine, plus 20 p m o l / k g per min neurotensin was calculated using one-way analysis of variance to be equivalent to the response to 204 p m o l / k g per min of peptide histidine methionine alone (95% confidence limits of 85-461 p m o l / k g per min) or 26 p m o l / k g per min of neurotensin alone (95% confidence limits of 15-55 p m o l / k g per rain), i.e. the response to both peptides together was not significantly different from the response to twice the dose of either peptide given alone.

4. Discussion

Neuromedin-N stimulated net secretion of fluid into the small intestine of the anaesthetised rat with the same pattern of activity as neurotensin, although it was about 21 times less potent. Neurotensin-(8-13) was 33 times less potent than neurotensin. The pattern of activity of these peptides differed from that of peptide histidine methionine in that only peptide histidine methionine had any effect in the duodenum. Although neuromedin-N has been shown to increase pancreatic, mesenteric and portal blood flow and to stimulate pancreatic exocrine secretion (Sumi et al., 1987), its activity in these systems has not been compared that of neurotensin. The results presented here are, therefore, probably the first direct comparison of the effects of neuromedin-N and neurotensin in an in vivo situation. The 20-fold lower potency of neuromedin-N compared with neurotensin found in the present study is in agreement with the findings of others in other systems. Minamino et al. (1984) found that neuromedin-N contracted isolated guinea-pig ileum with an activity of about 15% of that of neurotensin, Checler et al. (1986) showed that neuromedin-N bound to brain neurokinin receptors with a 19-fold lower potency than neurotensin and Kanba and Richelson (1987) showed that

neuromedin-N was about 10 times less potent than neurotensin at releasing [3H]inositol phosphates and stimulating intracellular cyclic [3H] G M P formation in the neuroblastoma clone N1E115. The difference in these relative potencies could be due to a number of reasons such as a difference in the rate at which the two peptides are metabolised under different experimental condition. Alternatively it may indicate the existence of separate receptors for neuromedin-N and neurotensin or different subtypes of receptor or a species difference. In fact, it has been shown that there is a slight difference between rat and guineapig neurotensin receptors (Checler et al., 1982). In the present study, neurotensin-(8-13) was found to have only 3% of the activity of neurotensin. It has previously been shown in other systems that this C-terminal hexapeptide of neurotensin, neurotensin-(8-13) is the shortest neurotensin analogue retaining the full spectrum of the activity of neurotensin itself (Carraway and Leeman, 1987; Granier et al., 1982). The relative potency of neurotensin-(8-13) in these studies, however, varied considerably from 15% of that of neurotensin for binding to rat brain synaptic membranes (Granier et al., 1982) to 350% of the activity of neurotensin for stimulating cyclic [3H]GMP formation in N1E-115 neuroblastoma clone cells (Kanba and Richelson, 1987). It has been shown, however, the arginine residues at position 8 and 9 (which differ in neuromedin-N) are not, themselves, a pre-requisite for activity as either can be replaced by lysine without significant loss of activity. The activity is lost, however, if they are replaced with citrate indicating that positively charged residues are required at positions 8 and 9 for full activity (Granier et al., 1982). This suggests that neuromedin-N, like neurotensin-(8-13), is the minimum structure likely to retain the full spectrum of neurotensin activity. These results also suggest that the reduced activity of neuromedin-N to neurotensin in this system is due to its length and not the difference in amino acid sequence. While all four peptides tested caused an increase in net fluid secretion into the lumen of the small intestine of the anaesthetised rat, they have different patterns of activity. Peptide histidine

47

methionine was active in all regions while neurotensin, neuromedin-N and neurotensin-(8-13) were inactive in the duodenum but highly active in the jejunum and ileum. It is interesting to note that these findings closely mimic the distribution of these peptides. Peptide histidine methionine (or the rat equivalent, peptide histidine isoleucine, which has a similar pattern of activity to peptide histidine methionine in this system (Spokes et al., 1988) is present in uniform quantities throughout the small intestine (Christofides et al., 1984), whereas neurotensin and neuromedin-N are present in low concentrations in the duodenum compared with their levels in the rest of the small bowel (Carraway and Mitra, 1987; Lee et al., 1987). These findings suggest that peptide histidine methionine and neurotensin/neuromedin-N act via different receptors (and possibly different mechanisms) to increase net fluid secretion into the rat small intestine. It was therefore decided to test low doses of peptide histidine methionine and neurotensin together to investigate the possibility that they might potentiate or antagonise each other. The response to both peptides administered together was found to be similar to that obtained by twice the dose of either peptide given alone, thus showing no evidence for any interaction between them. It is not possible to deduce from this data whether or not the post-receptor mechanism is the same for both peptides. The results presented here suggest that neuromedin-N could have a physiological role in the control of fluid movement across the gastrointestinal tract, although any such role is likely to be minor as neuromedin-N is far less potent than neurotensin.

References Carraway, R. and S.E. Leeman, 1976, Characterization of radioimmunoassayable neurotensin in the rat, J. Biol. Chem. 251, 7045. Carraway, R.E. and S.P. Mitra, 1987, The use of radioim-

munoassay to compare the tissue and subcellular distributions of neurotensin and neuromedin N in the cat, Endocrinology 120, 2092. Checler, F., L. Labbe., C. Granier, J. Van Rietschoten, P. Katabgi and J.P. Vincent, 1982, [TRPll]-Neurotensin and Xenopsin discriminate between rat and guinea-pig neurotensin receptors, Life Sci. 31, 1145. Checler, F., J.P. Vincent and P. Kitabgi, 1986, Neuromedin N: high affinity interaction with brain neurotensin receptors and rapid inactivation by brain synaptic peptidases European J. Pharmacol. 126, 239. Christofides, N.D., S.M. Polak and S.R. Bloom, 1984, Studies on the distribution of PHI in mammals, Peptides 5, 261. Dobner, P.R., D.L. Barber, L. Villa-Komaroff and C. McKiernan, 1987, Cloning and sequence analysis of CDNA for the canine neurotensin/neuromedin N precursor, Proc. Natl. Acad. Sci. 84, 3516. Ghiglione, M., N.D. Christofides, Y. Yiangou, L.O. Uttenthal and S.R. Bloom, 1982, PHI stimulates intestinal fluid secretion, Neuropeptides 3, 79. Granier, C., J. Van Rietschoten, P. Katabgi, C. Poustis and P. Freychet, 1982, Synthesis and characterization of neurotensin analogues for structure/activity relationship studies, European J. Biochem. 124, 117. Holzer, P., A. Bucsics, A. Saria and F. Lembeck, 1982, A study of the concentrations of substance P and neurotensin in the gastrointestinal tract of various mammals, Neuroscience 7, 2919. Kanba, K.S. and E. Richelson, 1987, Comparison of the stimulation of inositol phospholipid hydrolysis and of cell cyclic GMP formation by neurotensin, some of its analogs and neuromedin-N in neuroblastoma clone NIE-113, Biochem. Pharmacol. 36, 869. Lee, Y.C., J.A. Ball, D. Reece and S.R. Bloom, 1987, Neuromedin N: presence and chromatographic characterization in the rat, FEBS Lett. 220, 243. Minamino, N., K. Kangawa and H. Matsuo, 1984, Neuromedin N: a novel neurotensin-like peptide identified in porcine spinal cord, Biochem. Biophys. Res. Commun. 122, 542. Mitchenere, P., T.E. Adrian, R.M. Kirk and S.R. Bloom, 1981, Effect of gut regulatory peptides on intestinal luminal fluid in the rat, Life Sci. 29, 1563. Spokes, R.A., Y. Yiangou, B.J. Chrysanthou, M.P. Svensson Bowles and S.R. Bloom, 1988, Is VIP really the main contributor to the watery diarrhoea in patients with VIPsecreting tumours?, Clin. Sci. 74 (Suppl. 18) 69p. Sumi, S., K. Inoue, M. Kogire, R. Doi, K. Takaori, H. Yajima, T. Suzuki and T. Tobe, 1987, Effect of synthetic neuromedin°N, a novel neurotensin-like peptide, on exocrine pancreatic secretion and splanchnic blood flow in dogs, Neuropeptides 9, 247.