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Effects of neurotensin on isolated intestinal smooth muscles
European Journal o f Pharmacology, 50 (1978) 349--357 © Elsevier/North-Holland Biomedical Press
349
E F F E C T S OF N E U R O T E N S I N ON ISOLATED INTESTINAL SMOOTH MUSCLES PATRICK KITABGI and PIERRE FREYCHET Institut National de la Santd et de la Recherche Mddicale (I.N.S.E.R.M.), Groupe de Recherches sur les Hormones Polypeptidiques et la Physiopathologie Endocrinienne, U. 145, Facultd de Mddecine (Pasteur), 06034 Nice Cedex, France Received 30 December 1977, revised MS received 29 March 1978, accepted 27 April 1978
P. KITABGI and P. FREYCHET, Effects o f neurotensin on isolated intestinal smooth muscles, European J. Pharmacol. 50 (1978) 349--357. The effects of neurotensin were investigated in intestinal smooth muscle preparations. Neurotensin relaxed the rat ileum, and contracted the guinea-pig ileum and taenia. Neurotensin induced a biphasic response (relaxation followed by contraction) in the contracted guinea-pig ileum. In all systems, half-maximal effects were obtained with 4--5 nM neurotensin and maximal responses with 30--60 nM; tachyphylaxis occurred with higher concentrations. Tetrodotoxin did not affect the responses to neurotensin in the rat ileum and the guinea-pig taenia. Tetrodotoxin abolished the contraction or the contraction phase (but not the relaxation phase) of the biphasic response induced by neurotensin in the guinea-pig ileum. Atropine partially inhibited the neurotensininduced contraction in the guinea-pig ileum. These results suggest that neurotensin acts on intestinal smooth muscle both directly (relaxation of the rat and guinea-pig ileum, and contraction of the guinea-pig taenia) and through a nerve-mediated, partly cholinergic, process (contraction of the guinea-pig ileum). Neurotensin Tetrodotoxin
Intestinal smooth muscle
Guinea-pig ileum
1. Introduction Neurotensin (NT) is a tridecapeptide originally isolated from the hypothalamus (Carraway and Leeman, 1973). However, the major source of NT appears to be the gastrointestinal tract. Thus, about 90% of the total NT-immunoreactivity content of the rat has been found in the gastrointestinal tract (Carraway and Leeman, 1976). Furthermore, the NT-immunoreactivity has been purified from bovine intestine and demonstrated to be identical with hypothalamic NT (Kitabgi et al., 1976); it has also been shown that NT was present in cells of the mucosa of the small intestine (Orci et al., 1976; Sundler et al., 1977; Polak et al., 1977). In addition, NT possesses several pharmacological properties which involve the gastrointestinal tract as a target organ, e.g., inhibition of gastric acid secretion (Andersson et al., 1976), contraction of the guinea-
Rat ileum
Guinea-pig taenia
pig ileum, and relaxation of the rat d u o d e n u m (Carraway and Leeman, 1973). It thus appears that the gastrointestinal tract may play an important role both as a source of NT and as a target organ for this peptide. However, since the initial and brief report by Carraway and Leeman {1973) that NT was active on intestinal smooth muscle preparations, there have been few studies on the type and properties of this action. While the present work was in progress, two brief reports on the contractile activity of NT in the guinea-pig ileum have appeared (Segawa et al., 1977; RSkaeus et al., 1977). The present study is an attempt to characterize further the pharmacological effects of NT on isolated intestinal smooth muscle preparations. These effects were analyzed in the rat and guinea-pig ileum, and the guineapig taenia coll.
350 2. Materials and methods
2.1. Drugs Synthetic NT, substance P (SP) and bradykinin were purchased from Beckman. Other drugs used: acetylcholine chloride (ACh), histamine dihydrochloride, adrenaline hydrochloride, atropine sulphate and D,L-propranolol hydrochloride, were obtained from Sigma; phentolamine hydrochloride was from Ciba-Geigy; tetrodotoxin (TTX), Crystalline 3X, was from Sankyo. Chlorpheniramine maleate and nicotine hydrogen tartrate were gifts from Dr. P. Lapalus (Pharmacology department, Nice).
2.2. Rat isolated ileum preparation Rats (150--200 g) were decapitated; a segment of ileum 2--3 cm in length and situated 5 cm proximal to the ileo-caecal junction was used. The tissue was set up in a 10 ml organ bath containing Tyrode solution (mM) (136.8 NaC1; 2.7 KC1; 1 MgSO4.7H:O; 0.4 NaH2PO4 • H20; 11.9 NaHCO3; 3.6 CaCI:; 5.5 glucose) maintained at 37°C and gassed with 95% 02--5% CO2. The resting tension was 0.5 g and isometric contractions were recorded with a transducer (Apelab, Bagneux, France) connected to a potentiometric recorder (Servotrace, Sefram, Paris, France). After being set up the tissue was allowed to equilibrate for 45 min and was washed every 10 min. Dose--responses for NT were obtained by contracting the muscle with 2 gM ACh and by adding, 1 min later, NT cumulatively every 30 sec, so as to increase the concentration in the bath by a factor of 2 after each addition of the peptide. Cumulative dose-responses for adrenaline were obtained as described for NT. The effects of the ~-adrenergic blocking agent propranolol and the a-adrenergic blocking agent phentolamine were tested by adding these two drugs together at appropriate concentrations 5 min before starting a dose-response experiment for NT or adrenaline. In experiments designed to test the effect of
P. KITABGI, P. FREYCHET TTX, the tissue was exposed to the toxin at 0.3 pM for 5 to 30 min before the addition of other drugs.
2.3. Guinea-pig isolated ileum preparation Male guinea pig (300--400 g) were killed by a blow on the head; a segment 2--3 cm in length of the terminal portion of the ileum (5--6 cm proximal to the ileo-caecal junction) was used. The setting up of the muscle segment was identical to that described for rat muscle. Dose-responses for NT, SP and bradykinin were obtained by adding the agonist to the bath at 3 min intervals and at the concentrations indicated in the figures. The muscle was washed 1 min or 1.5 min after each addition of the peptide. When the biphasic activity of NT was studied, doseresponses were obtained as follows: the muscle was first contracted by a given dose of histamine and NT was added 20 sec later, when the contraction was maximal. The preparation was washed 1 min after the addition of the peptide. A new cycle was started 90s after the washout. Dose-responses for ACh and histamine were obtained by adding the drugs cumulatively every 30s so as to increase the concentration by a factor of 2 after each addition of the agonist. Experiments with atropine, and with the Hi-blocking antihistaminic, chlorpheniramine, were performed by adding these drugs to the ~reservoir of Tyrode solution. Experiments with TTX were carried out as described for the rat ileum.
2.4. Guinea-pig-isolated taenia preparation A single taenia was dissected from the caecum of the guinea-pig. Segments of taenia 2-3 cm in length were set up exactly as described above for the rat ileum. Cumulative dose-responses were obtained for NT, ACh and histamine by doubling the concentration of the drug after each addition to the bath. TTX was used as described for the rat and guineapig ileum. The guinea-pig taenia coli exhibits a spon-
NEUROTENSIN EFFECT ON INTESTINAL SMOOTH MUSCLE taneous activity which differs from one preparation to another depending on its tone; one can define the tone of a preparation as low, medium or high (Hobbiger et al., 1969). In order to facilitate the quantitative expression of the results, these are reported for preparations which exhibited a low basal tone t h r o u g h o u t the experiments. However, the results were qualitatively similar regardless of the tone of the preparation.
3. Results
3.1. Effects o f N T on the rat isolated ileum Neurotensin at concentrations up to 100 nM had no effect on this preparation when it had a basal, low tone. However, NT produced a quick relaxation of the muscle when added to a preparation with a spontaneously increased tone. To study the relaxing effect of NT, the preparation was first contracted with 2 pM ACh (a maximally effective dose) and a cumulative dose--response for NT was performed (fig. 1A); NT was effective in concentrations ranging from 1 to 120 nM. The relaxation occurred within seconds following the addition of the peptide and was complete in less than 30 sec. A maximal effect was obtained with 30--60 nM NT; higher concentrations failed to produce a greater response. Under these conditions, a desensitization of the muscle to NT was observed with the peptide at 120 riM. When the muscle was exposed to 2 gM ACh alone for 4--5 min (the time required to perform the cumulative dose-response to NT), a sustained contraction was observed indicating that no fading of the response to ACh interfered with the relaxing effect of NT. Unlike adrenaline (fig. 1B), NT was unable to achieve a full relaxation of the rat ileum contracted with a maximally effective dose of ACh. The maximal effect of NT was 3 8 + 4% (mean + S.E.M. of 3 experiments) of that achieved with adrenaline. The ECs0 for the relaxing effect of NT was 4.4 + 0.5 nM (mean -+ S.E.M. of 5 experiments); the
351
ECs0 for adrenaline was 430 + 90 nM (mean ± S.E.M. of 5 experiments). Propranolol at 6 pM together with phentolamine at 1.5 pM, concentrations which increased the ECs0 for adrenaline by a factor of 15, or TTX were without effect on the response of the rat ileum to NT (not shown).
3.2. Effects o f N T on the guinea-pig isolated ileum 3.2.1. Contracting effect o f N T In contrast to the rat ileum, the guinea-pig ileum was contracted when NT was added directly to the bath. A dose--response for the NT effect is shown in fig. 2A. The peptide was active at concentrations as low as 1--2 nM. The contraction occurred after 15--30 sec and was maximal 1 min following the addition of NT. Thereafter the tone of the preparation decreased spontaneously. A maximal effect was obtained for concentrations of NT around 3 0 n M . At higher concentrations, there was a desensitization of the muscle to NT. The dose--response curve for NT was compared to that for several other agonists. These results are summarized in table 1. The potency of NT was of the same order of magnitude as that of the other peptides, bradykinin and SP (3 times less and 3 times more potent than NT, respectively). However the effectiveness of NT was lower than that of the other agonists and was only 41% of that of histamine, a result similar to that obtained by RSkaeus et al. (1977) using the same preparation. Chlorpheniramine at 30 nM, a concentration which increased the ECs0 for histamine by a factor of 20, was without effect on the response of the muscle to NT (not shown). In contrast, atropine at 10 nM, a concentration which increased the ECs0 for ACh by a factor of 15, inhibited the maximal response of the guinea-pig ileum to NT by 50% (fig. 2). No further inhibition was observed with 50 nM atropine. The toxin TTX at 0.3 pM markedly inhibited the contraction produced by a maximally effective dose ( 3 0 n M ) of NT
352
P. K I T A B G I , P. F R E Y C H E T
ADRENALINE(nM) 30 a~6o
NT(nM) O95
ACh
~
2000~
~ v 3.8
~ ~
125
I
i
250
B
A
o.5
o.Is # i Imin
I
?o
i °°
i Imin
Fig. 1. C u m u l a t i v e d o s e - - r e s p o n s e s o f t h e relaxing effect of N T (A) a n d a d r e n a l i n e (B) o n t h e rat ileum cont r a c t e d b y ACh. Drugs were a d d e d t o give t h e final c o n c e n t r a t i o n s i n d i c a t e d o n t h e figure, e x p r e s s e d as nM. B, b u f f e r s o l u t i o n ; W, w a s h o u t .
TABLE 1 C o m p a r i s o n o f t h e p o t e n c y a n d effectiveness o f various agonists in t h e guinea-pig ileum. T h e values are m e a n s _+ S.E.M. of t h e n u m b e r of e x p e r i m e n t s given in parentheses. Agonist
ECs 0 (nM)
Relative p o t e n c y I
Effectiveness 2
Histamine ACh NT Bradykinin sP
170.0 + 23.0 (10) 4 3 . 5 + 5.1 (6) 4.5_+ 0.9 (4) 12.0 _+ 4.0 (3) 1.5 _+ 0.4 (3)
3 10 100 38 300
100 81-+5 41_+2 99 -+ 5 81 + 1
(3) (6) (3) (3)
1 F o r each agonist, t h e p o t e n c y relative to N T was calculated as [(ECs0 o f N T ) / ( E C s 0 o f a g o n i s t ) ] × 100. 2 F o r each agonist, effectiveness was calculated as [ ( m a x i m a l e f f e c t of a g o n i s t ) / ( m a x i m a l effect of h i s t a m i n e ) ] × 100.
NEUROTENSIN EFFECT ON INTESTINAL SMOOTH MUSCLE
353
ACh(nM) ,oo
A
250~1OO0
H (nM)
I
I ~ll, T ~'~'~/ !'~
I
NT(nM) 1.9
3.8 w
I t
75 w
15
30
w
w
2,, ~
60 w
w
I
1mill
ACh (nM) 9000 40~19000
H (nM)
B
200
f
NT(nM) 1.9
3.8
7.5
15
30
60
0"5gI 50C
25O
1251
I m,n
Fig. 2. Effect of atropine on the NT-induced guinea-pig ileum contraction. (A) Normal Tyrode solution; from left to right: cumulative dose--response for ACh; increasing doses of NT; single dose of histamine (H). All drugs were added to give the final concentrations indicated on the figure and expressed as nM. W, washout. (B) Tyrode solution containing 10 nM atropine; otherwise, same as (A). The same guinea-pig ileum preparation was used for (A) and (B).
(fig. 3). It should be noted that when the muscle was exposed to TTX for 10 min {right part of fig. 3); the tone of the preparation increased slightly, and when NT was subse-
quently added the tone immediately decreased to its initial level. This relaxation was followed by a small increase of the muscle tone which did not exceed the level attained during the
354
P. KITABGI, P. FREYCHET
NT(nM)
NT
30
NT
30
I
I
w
WW
I I
TTX
NT
30
I
NT
30
30
I
W
WWW
I TTX
I
II
I ,.
~5min O'SgI~
~ ' "
_
1rain Fig. 3. Effect of TTX (0.3 gM) on the NT-induced guinea-pig ileum contraction. The ileum was exposed to TTX for either 5 or 10 min before the addition of 30 nM NT. W, washout.
equilibration period with TTX. These results were repeatedly observed for various conditions of equilibration of the muscle with TTX. Whereas the toxin always abolished the contractile activity of NT, TTX had no effect on the contractions produced by ACh, histamine, SP or bradykinin.
NT 3O
I 1 H 5o
3.2.2. Biphasic effect of NT It was observed that the addition of NT to spontaneously contracted guinea-pig ileum was followed by a quick relaxation of the muscle and then by a contraction. This led us to study the effect of NT on a preparation which had been contracted by a given dose of histamine. As shown in fig. 4 {left), the addition of NT to a muscle contracted by 50 nM histamine produced a biphasic response, i.e., an immediate relaxation followed by a contraction. The magnitude of the response, measured as the height between the point of lowest tone and that of maximal tone after the addition of NT, was dose-dependent,,The ECs0 for this effect was 4.9 +- 0.7 nM (mean + S.E.M. of 8 experiments). Such biphasic responses were not observed with SP and bradykinin. The toxin TTX abolished the contraction phase of the NT-induced biphasic response, whereas it had no effect on the relaxation phase (fig. 4, right); in fact, the relaxation
H so NT
1
1
ao
/ TTX 20rain W
l
O'5gI
/
J
/'
i
[
I I'nrtt Fig. 4. Effect of TTX (0.3 pM) on the biphasic response induced by 30 nM NT in the guinea-pig ileum contracted by 50 nM histamine (H). The ileum was exposed for 20 min to TTX, during which time the recorder was stopped. W, washout.
NEUROTENSIN EFFECT ON INTESTINAL SMOOTH MUSCLE I00
Histamine~ •
/
f.p. 5C
.
/ /
=/=
& . . . . . . . _ A. NT 7
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355
-'s
[AGoNIsT], L.g M Fig. 5. Cumulative dose--response curves of the contraction of the guinea-pig taenia by NT (e -'), histamine (.. - - I ) and ACh (A A). The results are expressed as the percentage of the maximal contraction induced by histamine. Similarresults were obtained in two other experiments. Inset, NT-induced contraction in the guineapig taenia. Ordinate: % of contraction.
phase was often amplified in the muscle treated with TTX.
3.3. Effects o f N T on the guinea-pig isolated taenia coli Neurotensin strongly contracted the taenia coli preparation (fig. 5, inset). Cumulative dose--response curves were compared for NT, ACh and histamine (fig. 5). The peptide was effective at 1 to 30 nM, with an ECs0 of 4 + 1 nM (mean + S.E.M. of 3 experiments). A maximal contraction was obtained at 30 nM; at higher concentrations, desensitization occurred (not shown). These features of NT action on the guinea-pig taenia resembled those of the action on guinea-pig ileum. However, there were marked differences in other parameters of the responses to NT of these two preparations. (1) As shown in fig. 5 (inset), NT at 7.5 nM induced a quick contraction (within seconds) of the taenia. This contraction was maximal at 30 sec and was sustained for several minutes. (2) Only monophasic contractile responses of the taenia were observed
with NT, even when the muscle was first contracted by histamine (not shown). (3) The effectiveness of NT relative to that of histamine (fig. 5) was nearly twice as high in the taenia (74-+ 5%, mean + S.E.M. of 3 experiments) as in the ileum (41 + 2%, see table 1). (4) TTX at 0.3 pM had no effect on the NTinduced contraction in the taenia coli preparation; under the same conditions, the toxin largely prevented the contraction induced by 2pM nicotine (not shown).
4. Discussion The results presented here have shown that NT was active on various intestinal smooth muscle preparations: it relaxed the rat ileum, had a biphasic effect (relaxation followed by a contraction) on the guinea-pig ileum, and contracted the guinea-pig taenia. The dose-response curves of the effect of NT-in all three preparations displayed several c o m m o n features. They were obtained for the same range of NT concentrations (1--120 riM); the
356 ECs0 for NT was 4--5 nM in the three systems; the maximal effect was obtained for 30--60 nM; and tachyphylaxis occurred at higher concentrations of the peptide. These facts suggest that the receptors involved in the actions of NT have similar properties in the various preparations studied here. Neurotensin could exert its effects on isolated intestinal smooth muscle preparations in two ways: a direct action on the smooth muscle fiber (myogenic action); and -- o r an action on the intramural nerve plexuses (neurogenic action). In order to distinguish between these two possibilities, the toxin TTX, which selectively blocks the axonal conduction of nerve action potentials without affecting the smooth muscle fiber (Kao, 1966), was used. Neither the relaxing effect of NT on the rat ileum nor its contracting effect on the guinea-pig taenia were altered by TTX. This suggests that the effects of NT on these two preparations are of the myogenic type. Furthermore, the relaxing action of NT on the rat ileum was not affected by adrenergic-blocking agents, indicating that this effect of the peptide on this preparation was not exerted through the sympathetic innervation of the rat ileum. The effects of TTX on the NT-induced biphasic response in the guineapig ileum were somewhat more complex; the toxin almost completely blocked the contraction phase without affecting the initial relaxation phase. It thus appears that the relaxing effect of NT on the guinea-pig ileum results from a myogenic action. It should be noted that in the presence of TTX the guinea-pig ileum responded to NT in the same way (relaxation) as did the rat ileum. It is of interest that TTX blocked the contractile activity of NT in the guinea-pig ileum, indicating that this effect is of the neurogenic type. The observation that atropine significantly reduced the NT-induced contraction in the guinea-pig ileum is in agreement with this conclusion. Therefore, it seems that the neurogenic action of NT in this preparation is exerted, at least partly, through the parasym-
P. KITABGI, P. FREYCHET
pathetic innervation. These findings are particularly interesting in view of the possible role of NT as a neurotransmitter or neuromodulator, as suggested by its presence in the central nervous system (Carraway and Leeman, 1976; Kobayashi et al., 1977), by the existence of specific binding sites for NT in brain synaptic membranes (Kitabgi et al., 1977; Uhl et al., 1977; Lazarus et al., 1977), and by the hypothermic action of the peptide when injected intracisternally (Bissette et al., 1976; Brown et al., 1977). It should be noted that the contractile action of NT in the guinea-pig ileum does not appear to be mediated by histamine in contrast to what has been proposed for the actions of NT on blood pressure, vascular permeability, blood glucose and plasma insulin, glucagon, growth hormone and prolactin (Vale et al., 1977). Neurotensin has recently been reported to contract the guinea-pig ileum (Segawa et al., 1977; RSkaeus et al., 1977). However, it is clear from the present study that this contraction is a neurogenic action of the peptide, and that the myogenic effect of NT in the guinea-pig (as well as in the rat) ileum appears to be a relaxation rather than a contraction. This distinguishes NT from other peptides, e.g. SP, which contract the guinea-pig ileum by directly stimulating the smooth muscle fibers (Pernow, 1963); however, in contrast to its nerve-mediated contractile effect in the guinea-pig ileum, NT contracts the guinea-pig taenia through an apparently direct effect on this smooth muscle preparation. This effect resembles that recently described for the rat fundus strip (RSkaeus et al., 1977). Neurotensin is one member of the growing family of brain-gastrointestinal peptides, such as SP, somatostatin and the vasoactive intestinal polypeptide (VIP). The physiological role of these peptides is largely unknown, as well as the mechanisms of their pharmacological action. Further studies with the guineapig taenia and ileum should help to elucidate the mechanisms of myogenic and neurogenic actions of NT on smooth muscle.
NEUROTENSIN E F F E C T ON INTESTINAL SMOOTH MUSCLE Acknowledgements We thank Drs. P. Meyer and M. Worcel for helpful advice during this work; G. Visciano for excellent illustration work; and J. Duch for expert secretarial assistance. This work was supported in part by a grant from the Institut National de la Sant~ et de la Recherche M~dicale (A.T.P I.N.S.E.R.M. No. 16 7539).
References Andersson, S., D. Chang, K. Folkers and S. Rosell, 1976, Inhibition of gastric acid secretion in dogs by neurotensin, Life Sci. 19,367. Bissette, G., C.B. Nemeroff, P.T. Loosen, A.J. Prange, Jr. and M.A. Lipton, 1976, Hypothermia and intolerance to cold induced by intracisternal administration of the hypothalamic peptide neurotensin, Nature (London) 2 6 2 , 6 0 7 . Brown, M., J. Rivier and W. Vale, 1977, Bombesin: potent effects on thermoregulation in the rat, Science 1 9 6 , 9 9 2 . Carraway, R. and S.E. Leeman, 1973, The isolation of a new hypotensive peptide, neurotensin, from bovine hypothalami, J. Biol. Chem. 248, 6854. Carraway, R. and S.E. Leeman, 1976, Characterization of radioimmunoassayable neurotensin in the rat, J. Biol. Chem. 251, 7045. Hobbiger, F., F. Mitchelson and M.J. Rand, 1969, The actions of some cholinomimetic drugs on the isolated taenia of the guinea pig caecum, Brit. J. Pharmacol. 36, 53. Kao, C.Y., 1966, Tetrodotoxin, saxitonin and their significance in the study of excitation phenomena, Pharmacol. Rev. 18,997. Kitabgi, P., R. Carraway and S.E. Leeman, 1976, Isolation of a tridecapeptide from bovine intestinal tissue and its partial characterization as neurotensin, J. Biol. Chem. 251, 7053. Kitabgi, P., R. Carraway, J. Van Rietschoten, C. Gra-
357
nier, J.L. Morgat, A. Menez, S. Leeman and P. Freychet, 1977, Neurotensin: Specific binding to synaptic membranes from rat brain, Proc. Nat. Acad. Sci. U.S.A. 74, 1846. Kobayashi, R.M., M. Brown and W. Vale, 1977, Regional distribution of neurotensin and somatostatin in rat brain, Brain Res. 126, 584. Lazarus, L.H., M.R. Brown and M. Perrin, 1977, Distribution, localization and characteristics of neurotensin binding sites in the rat brain, Neuropharmacology 16,625. Orci, L., O. Baetens, C. Rufener, M. Brown, W. Vale and R. Guillemin, 1976, Evidence for immunoreactive neurotensin in dog intestinal mucosa, Life Sci. 19,559. Pernow, B., 1963, Pharmacology of Substance P, Ann. N.Y. Acad. Sci. 104,393. Polak, J.M., S.N. Sullivan, S.R. Bloom, A.M.J. Buchan, P. Facer, M.R. Brown and A.G.E. Pearse, 1977, Specific localisation of neurotensin to the N cell in human intestine by radioimmunoassay and immunocytochemistry, Nature 270,183. RSkaeus, •., E. Burcher, D. Chang, K. Folkers and S. Rosell, 1977, Actions of neurotensin and (Gln4)-neurotensin on isolated tissues, Acta Pharmacol. Toxicol. 4 1 , 1 4 1 . Segawa, T., M. Hosokawa, K. Kitagawa and H. Ya° gima, 1977, Contractile activity of synthetic neurotensin and related polypeptides on guineapig ileum, J. Pharm. Pharmacol. 29, 57. Sundler, F., R. K~/kanson, R.A. Hammer, J. Alumets, R. Carraway, S.E. Leeman and E.A. Zimmerman, 1977, Immuno-histochemical localization of neurotensin in endocrine cells of the gut, Cell Tiss. Res. 178,313. Uhl, G.R,, J.P. Bennett, Jr. and S.H. Snyder, 1977, Neurotensin, a central nervous system peptide: apparent receptor binding in brain membranes, Brain Res. 130, 299. Vale, W., C. Rivier and M. Brown, 1977, Regulatory peptides of the hypothalamus, Ann. Rev. Physiol. 39,473.
349
E F F E C T S OF N E U R O T E N S I N ON ISOLATED INTESTINAL SMOOTH MUSCLES PATRICK KITABGI and PIERRE FREYCHET Institut National de la Santd et de la Recherche Mddicale (I.N.S.E.R.M.), Groupe de Recherches sur les Hormones Polypeptidiques et la Physiopathologie Endocrinienne, U. 145, Facultd de Mddecine (Pasteur), 06034 Nice Cedex, France Received 30 December 1977, revised MS received 29 March 1978, accepted 27 April 1978
P. KITABGI and P. FREYCHET, Effects o f neurotensin on isolated intestinal smooth muscles, European J. Pharmacol. 50 (1978) 349--357. The effects of neurotensin were investigated in intestinal smooth muscle preparations. Neurotensin relaxed the rat ileum, and contracted the guinea-pig ileum and taenia. Neurotensin induced a biphasic response (relaxation followed by contraction) in the contracted guinea-pig ileum. In all systems, half-maximal effects were obtained with 4--5 nM neurotensin and maximal responses with 30--60 nM; tachyphylaxis occurred with higher concentrations. Tetrodotoxin did not affect the responses to neurotensin in the rat ileum and the guinea-pig taenia. Tetrodotoxin abolished the contraction or the contraction phase (but not the relaxation phase) of the biphasic response induced by neurotensin in the guinea-pig ileum. Atropine partially inhibited the neurotensininduced contraction in the guinea-pig ileum. These results suggest that neurotensin acts on intestinal smooth muscle both directly (relaxation of the rat and guinea-pig ileum, and contraction of the guinea-pig taenia) and through a nerve-mediated, partly cholinergic, process (contraction of the guinea-pig ileum). Neurotensin Tetrodotoxin
Intestinal smooth muscle
Guinea-pig ileum
1. Introduction Neurotensin (NT) is a tridecapeptide originally isolated from the hypothalamus (Carraway and Leeman, 1973). However, the major source of NT appears to be the gastrointestinal tract. Thus, about 90% of the total NT-immunoreactivity content of the rat has been found in the gastrointestinal tract (Carraway and Leeman, 1976). Furthermore, the NT-immunoreactivity has been purified from bovine intestine and demonstrated to be identical with hypothalamic NT (Kitabgi et al., 1976); it has also been shown that NT was present in cells of the mucosa of the small intestine (Orci et al., 1976; Sundler et al., 1977; Polak et al., 1977). In addition, NT possesses several pharmacological properties which involve the gastrointestinal tract as a target organ, e.g., inhibition of gastric acid secretion (Andersson et al., 1976), contraction of the guinea-
Rat ileum
Guinea-pig taenia
pig ileum, and relaxation of the rat d u o d e n u m (Carraway and Leeman, 1973). It thus appears that the gastrointestinal tract may play an important role both as a source of NT and as a target organ for this peptide. However, since the initial and brief report by Carraway and Leeman {1973) that NT was active on intestinal smooth muscle preparations, there have been few studies on the type and properties of this action. While the present work was in progress, two brief reports on the contractile activity of NT in the guinea-pig ileum have appeared (Segawa et al., 1977; RSkaeus et al., 1977). The present study is an attempt to characterize further the pharmacological effects of NT on isolated intestinal smooth muscle preparations. These effects were analyzed in the rat and guinea-pig ileum, and the guineapig taenia coll.
350 2. Materials and methods
2.1. Drugs Synthetic NT, substance P (SP) and bradykinin were purchased from Beckman. Other drugs used: acetylcholine chloride (ACh), histamine dihydrochloride, adrenaline hydrochloride, atropine sulphate and D,L-propranolol hydrochloride, were obtained from Sigma; phentolamine hydrochloride was from Ciba-Geigy; tetrodotoxin (TTX), Crystalline 3X, was from Sankyo. Chlorpheniramine maleate and nicotine hydrogen tartrate were gifts from Dr. P. Lapalus (Pharmacology department, Nice).
2.2. Rat isolated ileum preparation Rats (150--200 g) were decapitated; a segment of ileum 2--3 cm in length and situated 5 cm proximal to the ileo-caecal junction was used. The tissue was set up in a 10 ml organ bath containing Tyrode solution (mM) (136.8 NaC1; 2.7 KC1; 1 MgSO4.7H:O; 0.4 NaH2PO4 • H20; 11.9 NaHCO3; 3.6 CaCI:; 5.5 glucose) maintained at 37°C and gassed with 95% 02--5% CO2. The resting tension was 0.5 g and isometric contractions were recorded with a transducer (Apelab, Bagneux, France) connected to a potentiometric recorder (Servotrace, Sefram, Paris, France). After being set up the tissue was allowed to equilibrate for 45 min and was washed every 10 min. Dose--responses for NT were obtained by contracting the muscle with 2 gM ACh and by adding, 1 min later, NT cumulatively every 30 sec, so as to increase the concentration in the bath by a factor of 2 after each addition of the peptide. Cumulative dose-responses for adrenaline were obtained as described for NT. The effects of the ~-adrenergic blocking agent propranolol and the a-adrenergic blocking agent phentolamine were tested by adding these two drugs together at appropriate concentrations 5 min before starting a dose-response experiment for NT or adrenaline. In experiments designed to test the effect of
P. KITABGI, P. FREYCHET TTX, the tissue was exposed to the toxin at 0.3 pM for 5 to 30 min before the addition of other drugs.
2.3. Guinea-pig isolated ileum preparation Male guinea pig (300--400 g) were killed by a blow on the head; a segment 2--3 cm in length of the terminal portion of the ileum (5--6 cm proximal to the ileo-caecal junction) was used. The setting up of the muscle segment was identical to that described for rat muscle. Dose-responses for NT, SP and bradykinin were obtained by adding the agonist to the bath at 3 min intervals and at the concentrations indicated in the figures. The muscle was washed 1 min or 1.5 min after each addition of the peptide. When the biphasic activity of NT was studied, doseresponses were obtained as follows: the muscle was first contracted by a given dose of histamine and NT was added 20 sec later, when the contraction was maximal. The preparation was washed 1 min after the addition of the peptide. A new cycle was started 90s after the washout. Dose-responses for ACh and histamine were obtained by adding the drugs cumulatively every 30s so as to increase the concentration by a factor of 2 after each addition of the agonist. Experiments with atropine, and with the Hi-blocking antihistaminic, chlorpheniramine, were performed by adding these drugs to the ~reservoir of Tyrode solution. Experiments with TTX were carried out as described for the rat ileum.
2.4. Guinea-pig-isolated taenia preparation A single taenia was dissected from the caecum of the guinea-pig. Segments of taenia 2-3 cm in length were set up exactly as described above for the rat ileum. Cumulative dose-responses were obtained for NT, ACh and histamine by doubling the concentration of the drug after each addition to the bath. TTX was used as described for the rat and guineapig ileum. The guinea-pig taenia coli exhibits a spon-
NEUROTENSIN EFFECT ON INTESTINAL SMOOTH MUSCLE taneous activity which differs from one preparation to another depending on its tone; one can define the tone of a preparation as low, medium or high (Hobbiger et al., 1969). In order to facilitate the quantitative expression of the results, these are reported for preparations which exhibited a low basal tone t h r o u g h o u t the experiments. However, the results were qualitatively similar regardless of the tone of the preparation.
3. Results
3.1. Effects o f N T on the rat isolated ileum Neurotensin at concentrations up to 100 nM had no effect on this preparation when it had a basal, low tone. However, NT produced a quick relaxation of the muscle when added to a preparation with a spontaneously increased tone. To study the relaxing effect of NT, the preparation was first contracted with 2 pM ACh (a maximally effective dose) and a cumulative dose--response for NT was performed (fig. 1A); NT was effective in concentrations ranging from 1 to 120 nM. The relaxation occurred within seconds following the addition of the peptide and was complete in less than 30 sec. A maximal effect was obtained with 30--60 nM NT; higher concentrations failed to produce a greater response. Under these conditions, a desensitization of the muscle to NT was observed with the peptide at 120 riM. When the muscle was exposed to 2 gM ACh alone for 4--5 min (the time required to perform the cumulative dose-response to NT), a sustained contraction was observed indicating that no fading of the response to ACh interfered with the relaxing effect of NT. Unlike adrenaline (fig. 1B), NT was unable to achieve a full relaxation of the rat ileum contracted with a maximally effective dose of ACh. The maximal effect of NT was 3 8 + 4% (mean + S.E.M. of 3 experiments) of that achieved with adrenaline. The ECs0 for the relaxing effect of NT was 4.4 + 0.5 nM (mean -+ S.E.M. of 5 experiments); the
351
ECs0 for adrenaline was 430 + 90 nM (mean ± S.E.M. of 5 experiments). Propranolol at 6 pM together with phentolamine at 1.5 pM, concentrations which increased the ECs0 for adrenaline by a factor of 15, or TTX were without effect on the response of the rat ileum to NT (not shown).
3.2. Effects o f N T on the guinea-pig isolated ileum 3.2.1. Contracting effect o f N T In contrast to the rat ileum, the guinea-pig ileum was contracted when NT was added directly to the bath. A dose--response for the NT effect is shown in fig. 2A. The peptide was active at concentrations as low as 1--2 nM. The contraction occurred after 15--30 sec and was maximal 1 min following the addition of NT. Thereafter the tone of the preparation decreased spontaneously. A maximal effect was obtained for concentrations of NT around 3 0 n M . At higher concentrations, there was a desensitization of the muscle to NT. The dose--response curve for NT was compared to that for several other agonists. These results are summarized in table 1. The potency of NT was of the same order of magnitude as that of the other peptides, bradykinin and SP (3 times less and 3 times more potent than NT, respectively). However the effectiveness of NT was lower than that of the other agonists and was only 41% of that of histamine, a result similar to that obtained by RSkaeus et al. (1977) using the same preparation. Chlorpheniramine at 30 nM, a concentration which increased the ECs0 for histamine by a factor of 20, was without effect on the response of the muscle to NT (not shown). In contrast, atropine at 10 nM, a concentration which increased the ECs0 for ACh by a factor of 15, inhibited the maximal response of the guinea-pig ileum to NT by 50% (fig. 2). No further inhibition was observed with 50 nM atropine. The toxin TTX at 0.3 pM markedly inhibited the contraction produced by a maximally effective dose ( 3 0 n M ) of NT
352
P. K I T A B G I , P. F R E Y C H E T
ADRENALINE(nM) 30 a~6o
NT(nM) O95
ACh
~
2000~
~ v 3.8
~ ~
125
I
i
250
B
A
o.5
o.Is # i Imin
I
?o
i °°
i Imin
Fig. 1. C u m u l a t i v e d o s e - - r e s p o n s e s o f t h e relaxing effect of N T (A) a n d a d r e n a l i n e (B) o n t h e rat ileum cont r a c t e d b y ACh. Drugs were a d d e d t o give t h e final c o n c e n t r a t i o n s i n d i c a t e d o n t h e figure, e x p r e s s e d as nM. B, b u f f e r s o l u t i o n ; W, w a s h o u t .
TABLE 1 C o m p a r i s o n o f t h e p o t e n c y a n d effectiveness o f various agonists in t h e guinea-pig ileum. T h e values are m e a n s _+ S.E.M. of t h e n u m b e r of e x p e r i m e n t s given in parentheses. Agonist
ECs 0 (nM)
Relative p o t e n c y I
Effectiveness 2
Histamine ACh NT Bradykinin sP
170.0 + 23.0 (10) 4 3 . 5 + 5.1 (6) 4.5_+ 0.9 (4) 12.0 _+ 4.0 (3) 1.5 _+ 0.4 (3)
3 10 100 38 300
100 81-+5 41_+2 99 -+ 5 81 + 1
(3) (6) (3) (3)
1 F o r each agonist, t h e p o t e n c y relative to N T was calculated as [(ECs0 o f N T ) / ( E C s 0 o f a g o n i s t ) ] × 100. 2 F o r each agonist, effectiveness was calculated as [ ( m a x i m a l e f f e c t of a g o n i s t ) / ( m a x i m a l effect of h i s t a m i n e ) ] × 100.
NEUROTENSIN EFFECT ON INTESTINAL SMOOTH MUSCLE
353
ACh(nM) ,oo
A
250~1OO0
H (nM)
I
I ~ll, T ~'~'~/ !'~
I
NT(nM) 1.9
3.8 w
I t
75 w
15
30
w
w
2,, ~
60 w
w
I
1mill
ACh (nM) 9000 40~19000
H (nM)
B
200
f
NT(nM) 1.9
3.8
7.5
15
30
60
0"5gI 50C
25O
1251
I m,n
Fig. 2. Effect of atropine on the NT-induced guinea-pig ileum contraction. (A) Normal Tyrode solution; from left to right: cumulative dose--response for ACh; increasing doses of NT; single dose of histamine (H). All drugs were added to give the final concentrations indicated on the figure and expressed as nM. W, washout. (B) Tyrode solution containing 10 nM atropine; otherwise, same as (A). The same guinea-pig ileum preparation was used for (A) and (B).
(fig. 3). It should be noted that when the muscle was exposed to TTX for 10 min {right part of fig. 3); the tone of the preparation increased slightly, and when NT was subse-
quently added the tone immediately decreased to its initial level. This relaxation was followed by a small increase of the muscle tone which did not exceed the level attained during the
354
P. KITABGI, P. FREYCHET
NT(nM)
NT
30
NT
30
I
I
w
WW
I I
TTX
NT
30
I
NT
30
30
I
W
WWW
I TTX
I
II
I ,.
~5min O'SgI~
~ ' "
_
1rain Fig. 3. Effect of TTX (0.3 gM) on the NT-induced guinea-pig ileum contraction. The ileum was exposed to TTX for either 5 or 10 min before the addition of 30 nM NT. W, washout.
equilibration period with TTX. These results were repeatedly observed for various conditions of equilibration of the muscle with TTX. Whereas the toxin always abolished the contractile activity of NT, TTX had no effect on the contractions produced by ACh, histamine, SP or bradykinin.
NT 3O
I 1 H 5o
3.2.2. Biphasic effect of NT It was observed that the addition of NT to spontaneously contracted guinea-pig ileum was followed by a quick relaxation of the muscle and then by a contraction. This led us to study the effect of NT on a preparation which had been contracted by a given dose of histamine. As shown in fig. 4 {left), the addition of NT to a muscle contracted by 50 nM histamine produced a biphasic response, i.e., an immediate relaxation followed by a contraction. The magnitude of the response, measured as the height between the point of lowest tone and that of maximal tone after the addition of NT, was dose-dependent,,The ECs0 for this effect was 4.9 +- 0.7 nM (mean + S.E.M. of 8 experiments). Such biphasic responses were not observed with SP and bradykinin. The toxin TTX abolished the contraction phase of the NT-induced biphasic response, whereas it had no effect on the relaxation phase (fig. 4, right); in fact, the relaxation
H so NT
1
1
ao
/ TTX 20rain W
l
O'5gI
/
J
/'
i
[
I I'nrtt Fig. 4. Effect of TTX (0.3 pM) on the biphasic response induced by 30 nM NT in the guinea-pig ileum contracted by 50 nM histamine (H). The ileum was exposed for 20 min to TTX, during which time the recorder was stopped. W, washout.
NEUROTENSIN EFFECT ON INTESTINAL SMOOTH MUSCLE I00
Histamine~ •
/
f.p. 5C
.
/ /
=/=
& . . . . . . . _ A. NT 7
f •/
//
./J-/ -
;8
-
;7
-'6
5nM
io1n.'
~ACh
•/ /
I -9
/
355
-'s
[AGoNIsT], L.g M Fig. 5. Cumulative dose--response curves of the contraction of the guinea-pig taenia by NT (e -'), histamine (.. - - I ) and ACh (A A). The results are expressed as the percentage of the maximal contraction induced by histamine. Similarresults were obtained in two other experiments. Inset, NT-induced contraction in the guineapig taenia. Ordinate: % of contraction.
phase was often amplified in the muscle treated with TTX.
3.3. Effects o f N T on the guinea-pig isolated taenia coli Neurotensin strongly contracted the taenia coli preparation (fig. 5, inset). Cumulative dose--response curves were compared for NT, ACh and histamine (fig. 5). The peptide was effective at 1 to 30 nM, with an ECs0 of 4 + 1 nM (mean + S.E.M. of 3 experiments). A maximal contraction was obtained at 30 nM; at higher concentrations, desensitization occurred (not shown). These features of NT action on the guinea-pig taenia resembled those of the action on guinea-pig ileum. However, there were marked differences in other parameters of the responses to NT of these two preparations. (1) As shown in fig. 5 (inset), NT at 7.5 nM induced a quick contraction (within seconds) of the taenia. This contraction was maximal at 30 sec and was sustained for several minutes. (2) Only monophasic contractile responses of the taenia were observed
with NT, even when the muscle was first contracted by histamine (not shown). (3) The effectiveness of NT relative to that of histamine (fig. 5) was nearly twice as high in the taenia (74-+ 5%, mean + S.E.M. of 3 experiments) as in the ileum (41 + 2%, see table 1). (4) TTX at 0.3 pM had no effect on the NTinduced contraction in the taenia coli preparation; under the same conditions, the toxin largely prevented the contraction induced by 2pM nicotine (not shown).
4. Discussion The results presented here have shown that NT was active on various intestinal smooth muscle preparations: it relaxed the rat ileum, had a biphasic effect (relaxation followed by a contraction) on the guinea-pig ileum, and contracted the guinea-pig taenia. The dose-response curves of the effect of NT-in all three preparations displayed several c o m m o n features. They were obtained for the same range of NT concentrations (1--120 riM); the
356 ECs0 for NT was 4--5 nM in the three systems; the maximal effect was obtained for 30--60 nM; and tachyphylaxis occurred at higher concentrations of the peptide. These facts suggest that the receptors involved in the actions of NT have similar properties in the various preparations studied here. Neurotensin could exert its effects on isolated intestinal smooth muscle preparations in two ways: a direct action on the smooth muscle fiber (myogenic action); and -- o r an action on the intramural nerve plexuses (neurogenic action). In order to distinguish between these two possibilities, the toxin TTX, which selectively blocks the axonal conduction of nerve action potentials without affecting the smooth muscle fiber (Kao, 1966), was used. Neither the relaxing effect of NT on the rat ileum nor its contracting effect on the guinea-pig taenia were altered by TTX. This suggests that the effects of NT on these two preparations are of the myogenic type. Furthermore, the relaxing action of NT on the rat ileum was not affected by adrenergic-blocking agents, indicating that this effect of the peptide on this preparation was not exerted through the sympathetic innervation of the rat ileum. The effects of TTX on the NT-induced biphasic response in the guineapig ileum were somewhat more complex; the toxin almost completely blocked the contraction phase without affecting the initial relaxation phase. It thus appears that the relaxing effect of NT on the guinea-pig ileum results from a myogenic action. It should be noted that in the presence of TTX the guinea-pig ileum responded to NT in the same way (relaxation) as did the rat ileum. It is of interest that TTX blocked the contractile activity of NT in the guinea-pig ileum, indicating that this effect is of the neurogenic type. The observation that atropine significantly reduced the NT-induced contraction in the guinea-pig ileum is in agreement with this conclusion. Therefore, it seems that the neurogenic action of NT in this preparation is exerted, at least partly, through the parasym-
P. KITABGI, P. FREYCHET
pathetic innervation. These findings are particularly interesting in view of the possible role of NT as a neurotransmitter or neuromodulator, as suggested by its presence in the central nervous system (Carraway and Leeman, 1976; Kobayashi et al., 1977), by the existence of specific binding sites for NT in brain synaptic membranes (Kitabgi et al., 1977; Uhl et al., 1977; Lazarus et al., 1977), and by the hypothermic action of the peptide when injected intracisternally (Bissette et al., 1976; Brown et al., 1977). It should be noted that the contractile action of NT in the guinea-pig ileum does not appear to be mediated by histamine in contrast to what has been proposed for the actions of NT on blood pressure, vascular permeability, blood glucose and plasma insulin, glucagon, growth hormone and prolactin (Vale et al., 1977). Neurotensin has recently been reported to contract the guinea-pig ileum (Segawa et al., 1977; RSkaeus et al., 1977). However, it is clear from the present study that this contraction is a neurogenic action of the peptide, and that the myogenic effect of NT in the guinea-pig (as well as in the rat) ileum appears to be a relaxation rather than a contraction. This distinguishes NT from other peptides, e.g. SP, which contract the guinea-pig ileum by directly stimulating the smooth muscle fibers (Pernow, 1963); however, in contrast to its nerve-mediated contractile effect in the guinea-pig ileum, NT contracts the guinea-pig taenia through an apparently direct effect on this smooth muscle preparation. This effect resembles that recently described for the rat fundus strip (RSkaeus et al., 1977). Neurotensin is one member of the growing family of brain-gastrointestinal peptides, such as SP, somatostatin and the vasoactive intestinal polypeptide (VIP). The physiological role of these peptides is largely unknown, as well as the mechanisms of their pharmacological action. Further studies with the guineapig taenia and ileum should help to elucidate the mechanisms of myogenic and neurogenic actions of NT on smooth muscle.
NEUROTENSIN E F F E C T ON INTESTINAL SMOOTH MUSCLE Acknowledgements We thank Drs. P. Meyer and M. Worcel for helpful advice during this work; G. Visciano for excellent illustration work; and J. Duch for expert secretarial assistance. This work was supported in part by a grant from the Institut National de la Sant~ et de la Recherche M~dicale (A.T.P I.N.S.E.R.M. No. 16 7539).
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357
nier, J.L. Morgat, A. Menez, S. Leeman and P. Freychet, 1977, Neurotensin: Specific binding to synaptic membranes from rat brain, Proc. Nat. Acad. Sci. U.S.A. 74, 1846. Kobayashi, R.M., M. Brown and W. Vale, 1977, Regional distribution of neurotensin and somatostatin in rat brain, Brain Res. 126, 584. Lazarus, L.H., M.R. Brown and M. Perrin, 1977, Distribution, localization and characteristics of neurotensin binding sites in the rat brain, Neuropharmacology 16,625. Orci, L., O. Baetens, C. Rufener, M. Brown, W. Vale and R. Guillemin, 1976, Evidence for immunoreactive neurotensin in dog intestinal mucosa, Life Sci. 19,559. Pernow, B., 1963, Pharmacology of Substance P, Ann. N.Y. Acad. Sci. 104,393. Polak, J.M., S.N. Sullivan, S.R. Bloom, A.M.J. Buchan, P. Facer, M.R. Brown and A.G.E. Pearse, 1977, Specific localisation of neurotensin to the N cell in human intestine by radioimmunoassay and immunocytochemistry, Nature 270,183. RSkaeus, •., E. Burcher, D. Chang, K. Folkers and S. Rosell, 1977, Actions of neurotensin and (Gln4)-neurotensin on isolated tissues, Acta Pharmacol. Toxicol. 4 1 , 1 4 1 . Segawa, T., M. Hosokawa, K. Kitagawa and H. Ya° gima, 1977, Contractile activity of synthetic neurotensin and related polypeptides on guineapig ileum, J. Pharm. Pharmacol. 29, 57. Sundler, F., R. K~/kanson, R.A. Hammer, J. Alumets, R. Carraway, S.E. Leeman and E.A. Zimmerman, 1977, Immuno-histochemical localization of neurotensin in endocrine cells of the gut, Cell Tiss. Res. 178,313. Uhl, G.R,, J.P. Bennett, Jr. and S.H. Snyder, 1977, Neurotensin, a central nervous system peptide: apparent receptor binding in brain membranes, Brain Res. 130, 299. Vale, W., C. Rivier and M. Brown, 1977, Regulatory peptides of the hypothalamus, Ann. Rev. Physiol. 39,473.