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[TRP11]-Neurotensin and xenopsin discriminate between rat and guinea-pig neurotensin receptors
Life Sciences, Vol. 31, pp. 1145-1150 Printed in the U.S.A.
Pergamon Press
"~[TRpII]-NEUROTENSIN AND XENOPSIN DISCRIMINATE BETWEEN RAT AND GUINEA-PIG NEUROTENSIN RECEPTORS Frederic Checler I, Catherine Labb~ *I, Claude Granler*, Jurphaas van Rietschoten*, Patrick Kitabgl 2, and Jean-Pierre Vincent. Cent re de Biochimle du Centre National de la Recherche Scientifique (CNRS), Facult~ des Sciences, Parc Valrose, 06034 Nice C~dex, France. *INSEAM SC I0, Laboratoire de Biochimie, Facult~ de M~decine-Nord, Boulevard Pierre Dramard, 13326 Mars eille C~dex, France. (Received in final form July i, 1982) S u m ~ r~ The bindin~ and biological activities of neurotensin and two analogues, [Trp l]-neurotensln and xenopsln, in which a tryptophan replaces the neurotensin residue Tyr 11, were compared in rat and gulnea-plg. The binding activity of the three peptldes was measured as their ability to inhibit the binding of [3H]neurotensin to rat and guinea-pig brain synaptlc membranes. Their biological activities were measured as their effects on the contractility of rat and guineapig ileal smooth muscle preparations. In binding as well as biological assays, it was found that [Trpll]-neurotensin and xenopsin were as potent as neurotensin in the rat. In contrast, the two analogues were about i0 times less potent than neurotensin in the guinea-plg. These findings reveal differences between rat and guinea-pig neurotens in receptors. Such species-related differences in neurotensin receptors should be considered when comparing the activity of neurotens in analogues in assays using tissue preparations from various animal species.
After the tridecapeptide neurotensln (NT) was isolated by Carraway and Leeman (I), the availability of NT synthetic partial sequences led to the observation by these authors that the C-termlnal hexapeptide (NT 8-13) was the minimal sequence required for the full expression of NT biological activity (2). This was later confirmed by several studies in which the activities of NT partial sequences and a number of NT analogues were measured in radloreceptor assays (3-5), in vivo (6,7) and in vitro (5,8-13) bloassays. Although most of these analogues displayed similar potencies in a variety of in vitro assays (5,11-13), there was one noticeable exception, the analogue [TrpI~]-NT in which a tryptophyl residue has been substituted for the tyrosyl residue at position II in the NT molecule (Table I). The biological activity of this analogue was tested in four assay systems, i.e. rat stomach strip, coronary vessels, portal vein, and gulnea-pig atria (11-13). In all the preparations derived from rat tissues, [TrplI]-NT was found to be slightly more active than NT (11-13). In contrast, [TrplI]-NT had only 3.2% of the activity of NT in isolated gulnea-plg atria (II). These observa-
Recipients of a fellowship from the D~l~gation G~n~rale ~ la Recherche Sclentiflque et Technique ( D ~ S T , Paris). To whom all correspondence and reprint requests should be addressed. 0024-3205/82/i i 1145-06503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
1146
Rat and Gulnea-plg Neurotensin Receptors
Vol. 31, No. Ii, 1982
tlons suggested to us that NT receptors in rat might be different from those in gulnea-plg with regard to thelr specificity towards NT and [Trpll]-NT. Furthermore this hypothesis is supported by a report (14) which compared the activity of xenopsin (XE) and NT in several bioassays. Xenopsln is an octapeptlde isolated from frog skin (15) whose C-terminal hexapeptlde amino acid sequence strongly resembles that of NT (Table I). In the positlon corresponding to Tyr II for NT, XE possesses a tryptophyl residue. It was found that XE and NT were approximately equipotent in in vltro bioassays with rat preparations (isolated stomach strip and duodenum). In contrast, XE was 5-10 times less potent than NT in assays with guinea-pig bioassays (isolated ileum and atrium) (14). The present study was designed to test the hypothesis that rat and gulnea~ pig receptors have a different specificity for NT congeners with a tryptophan substituted for Tyr II. NT, [TrplI]-NT and XE were compared for their ability to inhibit the binding of [SH]NT to rat and guinea-plg brain synaptic membranes, and for their effect on the contractile activity of rat and guinea-plg ileum.
TABLE I Amino-Acid Sequences of NT, [TrplI]-NT and XE Pep t id e
1
2
3
4
5
6
7
8
9
I0
ii
12
13
NT
pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-lle-Leu-OH
[ Trpl I ]-NT
pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Trp-lle-Leu-OH
XE
pGlu-Gly-Lys-Arg-Pro-Trp-lle-Leu-OH Materials and Methods
Peptides. The amlno-acid sequences of NT, [TrplI]-NT and XE are shown in Table I. Synthetic XE was purchased from Beckman Bioproducts Department (Geneva, Switzerland). NT and [TrplI]-NT were synthesized by three of us (C.L., C.G. and J.V.R.). The synthesis of NT has been previously reported (16). [TrplI]-NT was synthesized following the same general procedure as that described for NT. Details of this synthesis will be given elsewhere (manuscript in preparation). [3H]NT (specific activity 65 Ci/mmol) was prepared by Jean-Louis Morgat (Centre d'Etudes Nucl~aires de Saclay, Gif-sur-Yvette, France) as previously described (4). [3H]NT (specific activity 61 Ci/mmol) was also purchased from New England Nuclear (Paris, France). Radloreceptor assays. Assay conditions and properties of [3H]NT binding to rat brain synaptic membranes have been described elsewhere (4). Briefly,[3H]NT at 2 nM was incubated for 30 min at 24°C in 250 ~i of 50 mM Tris-HCl, pH 7.5, containing 0.2% bovine serLml albumin, 0.4 mg/ml membrane protein, and varying concentrations of unlabeled peptides. Bound peptide was separated from free peptide by filtration on Milllpore filters (EGWP 0.2 ~m). The specific binding was calculated as the difference between the amount of radioactivity bound in the absence and in the presence of an excess (i ~M) of unlabeled NT. The same assay conditions were used for measuring the binding of NT, [TrplI]-NT and XE to guineapig brain synaptic membranes. These membranes were prepared from whole guineapig brain (minus cerebellum) according to the method used for preparing rat brain membranes (4). Preliminary experiments indicated that the binding of [=HINT (2 nM) to guinea-pig brain membranes measured as a function of time, reached a plateau by 20 min (not shown). At the plateau, total binding represented about 1% of the radioactivity (30,000 cpm) present in the incubation medium and nonspecific binding amounted to 10% of total binding. These values are similar to those obtained with rat brain membranes (4).
Vol. 31, No. II, 1982
Rat and Guinea-pig Neurotensin Receptors
1147
Biological assays. NT has previously been shown to contract isolated longitudinal smooth muscle strips from the guinea-pig ileum (17,18). The contractions result from the stimulation by NT of intestinal cholinergic excitatory nerves, and are enhanced in the presence of the ant icholinesterase agent neostigmine (18). This has been the basis of a convenient bloassay for NT and related peptides (5,18). Briefly, longitudinal smooth muscle strips (3-4 cm in length) were dissected from the guinea-pig ileum and set up in a I0 ml organ bath for isometric tension recording. After equilibration of the preparations at 37°C in Tyrode solution, pH 7.4, containing 0.i ~M neosti~mine methylsulfate, non-cumulatlve concentratlon-response curves for NT, [Trp~I]-NT and XE were obtained as previously described (18). NT has been shown to relax the longitudinal muscle of rat ileal segments that were previously contracted by acetylcholine (19). In the present study, longitudinal smooth muscle strips from rat ileum were obtained using the procedure previously reported for guinea-plg preparations (18). The rat ileal strips (3-4 cm in length) were set up for tension recording in normal Tyrode solution. After equilibration of the preparations (60-90 min), concentration-response curves for the relaxing effect of NT, [TrpII]-NT and XE were obtained as follows : the muscle preparation was contracted with 0.5 ~M acetylcholine chloride and varying concentrations of a given peptide were added i min later. The preparation was washed I min after the addition of peptide and a new cycle was started 3-6 min after the washout. The peptide-induced relaxation was measured as the maximal drop in tension which occurred within 10-15 sec following the addition of peptide. During this 10-15 sec period, the fading of the contraction elicited by acetylcholine was negligible. Muscle preparations were allowed to rest for 15-20 min after a complete concentration-response curve was obtained. Results Concentratlon-response curves for the inhibition of [3HINT binding by NT, [Trp11] NT and XE, to rat and guinea-pig brain synaptic membranes are shown in Fig. i. The three peptides were virtually equipotent in the rat radioreceptor assay (Fig. I, left). By contrast, in the guinea-plg binding assay [Trp11]-NT and XE were much less potent than NT (Fig. i, right). Table II shows that the ICs0 value for NT was similar, about 4 nM, in both binding assays, whereas the ICs0 values for [Trp 11]-NT and XE were I0 times higher in the guinea-pig than in the rat radioreceptor assay. TABLE II Comparison of the Binding and Biological Activities of NT, Rat and Guinea-Pig Rat Peptide
NT [ Trpl I ] -NT XE
Binding assay ICs0 (nM) 4.0 4.3 4.0
Trp 11 -NT and XE in
Gulnea-pig Bioassay ECs0 (nM) 2.1 1.7 2.6
Binding assay ICs0 (nM) 3.7 40 46
Bioassay ECs0 (nM) 4.2 30 20
ICs0 (concentration of peptide that inhibits 50% of the binding of [3H]NT) and EC$0 (concentration of peptlde that produces half-maxlmal effect) were derived from Fig. 1 and 2, respectively. Concentratlon-response curves for the biological effect of NT, [TrpII]-NT and XE in rat and gulnea-plg ileum are shown in Fig. 2 (left) and Fig. 2 (right),
1148
Rat and Gulnea-plg Neurotensln Receptors
Vol. 31, No. 11, 1982
FIG. I Competitive inhibition by unlabeled NT (9~'Q), [Trp .I ]-NT (I~'~) and XE (A~4&) of [3H]NT binding to synaptic membranes from rat (left panel) and gulnea-plg (right panel) brain. Binding is expressed as the percentage of initial binding of [3H]NT, i.e. the binding of [3H]NT in the absence of unlabeled peptide. The non-speclfic binding has been subtracted. Points are the mean _+ SE from 7 and 3 experiments with unlabeled NT in the rat guinea-pig radioreceptor assays, respectively, and the mean from 3 and 2 experiments with [TrpII]-NT and XE in the rat and gulnea-plg binding assays, respectively. .
i i [m,~],~M
"~
-s
-7
-6
FIG. 2 Concentratlon-response curves for the effects of NT (9~'O), [TrpII]-NT ( m - l ) and XE (A--A) on the contractility of rat (left panel) and guineapig (right panel) ileum. Results are expressed as the percentage of the maximal effect induced by NT. Points are the mean _+ SE from 7, 4 and 4 experiments with NT, [Trpll]-NT and XE, respectively, in the rat assay, and from 7, 3 and 4 experiments with NT, 'ITrpII]-NT and XE, respectively, in the guinea-plg assay.
Vol. 31, No. ii, 1982
Rat and Guinea-pig Neurotensin Receptors
1149
respectively. The three peptides exhibited similar potency in the rat bioassay. In the guinea-pig bioassay, the ~otencies of [TrplI]-NT and XE were lower than that of NT. In both assays, [Trp I]-NT and XE were as effective as NT, indicating that these peptides behaved llke full agonists. ECs0 values derived from the curves in Fig. 2 are shown in Table II. The values were close to 2 nM for all three pe~tides in the rat bioassay. In the guinea-pig bloassay, the ECs0 values for [Trp I]-NT and XE were 5-7 times higher than that for NT (4.2 nM). The relative potency of [TrplI]-NT in the binding and biological assays described here are shown in Table III together with the potency values obtained by others in various i__~nvitro assays (13). Comparison among the values derived from 5 rat and 3 guinea-pig assay systems involving brain tissues as well as peripheral tissues such as gastro-intestlnal or vascular smooth muscle, and cardiac muscle shows that [TrplI]-NT had 93-130% of the activity of NT in all assays with rat preparations, and only 3-14% in assays with guinea-pig preparations. TABLE III Relative P~tency of ~Trp II]-NT in Binding and Biological Assays from Rat and Guinea-Pig-. Rat Synaptic Membranes
lleum
93
124
Stomach Strip 13~
Guinea-plg Coronary Vessels I0~
Portal Vein I15b
Synaptic Membranes 9.2
lleum
14
Atria
3.2 b
In all assays, potency values are expressed relative to NT taken as I00. Values derived from reference (13). Discussion The data presented here have clearly shown that, in the gulnea-pig, [Trp II ]-NT and XE are less active than NT. However, in the rat, the three peptldes are nearly equipotent in binding to NT receptors and in activating these receptors. Our results are in agreement with those of others (11-13) who have measured the activity of [Trp II ]-NT in several bloassays using rat and guinea-pig tissues (Table III). The present results also agree with the report (14) that XE was almost as potent as NT in the rat, whereas XE was 5-10 times less potent than NT in the gulnea-pig. Taken together, these observations show that, unlike NT receptors in rat, the receptors in guinea-plg discriminate between NT and NT analogues in which a tryptophan replaces the tyroslne normally present at position II of the NT molecule. This strongly suggests that NT receptors in the rat differ from those in the guinea-pig. However, it should be noted that a n~nber of NT partial sequences and analogues, for example [Phe II ]-NT, were shown to have similar potencies in various rat and gulnea-plg assays (5, 8-13). Therefore, it appears that rat and guinea-pig NT receptors are not grossly different but rather exhibit limited differences which can be detected with NT analogues bearing appropriate modifications. Whether such differences can be observed in other species and with other NT analogs is not known at present. However, this possibility should be considered when comparing the activity of NT analogues in bloassays and binding assays using tissue preparations from various animal species. Previous studies with NT analogues modified at position II of the HT amino acid sequence have revealed that Tyr 1 ! plays a crucial role in the binding of NT to its receptors (5) and for the expression of biological activity (5-7, 10-13). Furthermore, modifications of position II have led to two NT analogues ([D-Trp II ]NT and [Tyr(Me~ I ]-NT) which behave as partial NT antagonists in certain tissue preparations and as weak agonlsts in other preparations, thus suggesting some
1150
Rat and Gulnea-plg Neurotensln Receptors
Vol. 31, No. ii, 1982
degree of tissue-related differences among NT receptors (13), Finally, comparisons of the in vivo and in vitro activities of NT analogues with D-amlno acids at position II have suggested that Tyr II is involved in the inactivation of NT in brain tissues (5). These observations, together with the present study, suggest that appropriate modifications or substitutions at the key position II of the NT molecule may provide NT analogues with interesting properties such as NT antagonists, analogues resistant to degradation, and species- or tissuespecific analogues. Acknow led~ement s We thank Jean-Louls Morgat (Centre d'Etudes Nucl~alres de Saclay, Gif-sur-Yvette, France) for the gift of [3H]NT, Ellen Van Obberghen-Schilling for careful reading of the manuscript, and Catherine Roulinat for expert secretarial assistance. This work was supported in part by grant 81.E.0542 from the D~l~gation G~n~rale la Recherche Sclentifique et Technique (DGRST, Paris) and by the Fondation pour la Recherche M~dicale. References i. 2.
3.
4.
5.
6. 7. 8. 9. I0. II. 12. 13. 14. 15. 16. 17. 18. 19.
R. CARRAWAY and S.E. LEEMAN, J. Biol. Chem. 248 6854-6861 (1973). R. CARRAWAY and S.E. LEEY~AN, Peptides : Chemistry, Structure and Biolosy , R. Walter and J. Meienhofer eds., pp. 679-685, Ann Arbor Science Publishers, Ann Arbor, Michigan (1975). G.R. UHL, J.P. BENNETT and S.H. SNYDER, Brain Res. 130 299-313 (1977). P. KITABGI, R. CARRAWAY, J. VAN RIETSCHOTEN, C. GRANIER, J.L. MORGAT, A. MENEZ, S. LEEMAN and P. FREYCHET, Proc. Natl. Acad. Sci. USA 74 1846-1850 (1977). P. KITABGI, C. POUSTIS, C. GRANIER, J. VAN RIETSCHOTEN, J. RIVIER, J.L. MORGAT and P. FREYCHET, Mol. Pharmacol. 18 11-19 (1980). J. RIVIER, L.H. LAZARUS, M.H. FERRIN and M.R. BROWN, J. Med. Chem. 20 14091412 (1977). P.T. LOOSEN, C.B. N~EROFF, G. BISSETTE, G.B. BURNETT, A.J. PRANGE Jr and M.A. LIPTON, Neuropharmacology 17 109-113 (1978). T. SEGAWA, M. HOSOKAWA, K. KITAGAWA and H. YAJIMA, J. Pharm. Pharmacol. 29 57-58 (1977). K. KATAOKA, A. TANIGUCHI, H. SHIMIZU, K. SODA, S. OKUNO, H. YAJIMA and K. KITAGAWA, Brain Res. Bull. 3 555-557 (1978). R. QUIRION, D. REGOLI, F. RIOUX and S. ST-PIERRE, Br. J. Pharmacol. 68 8391 (1980). R. QUIRION, D. REGOLI, F. RIOUX and S. ST-PIERRE, Br. J. Pharmacol. 69 689692 (1980). R. QUIRION, F. RIOUX, D. REGOLI and S. ST-PIERRE, European J. Phannacol. 66 257-266 (1980). F. RIOUX, R. QUIRION, D. REGOLI, M.A. LEBLANC and S. ST-PIERRE, European J. Pharmacol. 66 273-279 (1980). K. ARAKI, S. TACHIBANA, Y. KATO and T. TAJIMA, Yakugaku Zasshi 99 466-470 (1979). K. ARAKI, S. TACHIBANA, M. UCHIYAMA, T. NAKAJIMA and T. YASUHARA, Chem. Pharm. Bull. 23 3132-3140 (1975). C. GRANIER, J. VAN RIETSCHOTEN, P. KITABGI, C. POUSTIS and P. FREYCHET, Eur. J. Biochem. 124 117-124 (1982). P. KITABGI and P. FREYCHET, European J. Pharmacol. 55 35-42 (1979). P. KITABGI and P. FREYCHET, European J. Pharmacol. 56 403-406 (1979). P. KITABGI and P. FREYCHET, European J. Pharmacol. 50 349-357 (1978).
Pergamon Press
"~[TRpII]-NEUROTENSIN AND XENOPSIN DISCRIMINATE BETWEEN RAT AND GUINEA-PIG NEUROTENSIN RECEPTORS Frederic Checler I, Catherine Labb~ *I, Claude Granler*, Jurphaas van Rietschoten*, Patrick Kitabgl 2, and Jean-Pierre Vincent. Cent re de Biochimle du Centre National de la Recherche Scientifique (CNRS), Facult~ des Sciences, Parc Valrose, 06034 Nice C~dex, France. *INSEAM SC I0, Laboratoire de Biochimie, Facult~ de M~decine-Nord, Boulevard Pierre Dramard, 13326 Mars eille C~dex, France. (Received in final form July i, 1982) S u m ~ r~ The bindin~ and biological activities of neurotensin and two analogues, [Trp l]-neurotensln and xenopsln, in which a tryptophan replaces the neurotensin residue Tyr 11, were compared in rat and gulnea-plg. The binding activity of the three peptldes was measured as their ability to inhibit the binding of [3H]neurotensin to rat and guinea-pig brain synaptlc membranes. Their biological activities were measured as their effects on the contractility of rat and guineapig ileal smooth muscle preparations. In binding as well as biological assays, it was found that [Trpll]-neurotensin and xenopsin were as potent as neurotensin in the rat. In contrast, the two analogues were about i0 times less potent than neurotensin in the guinea-plg. These findings reveal differences between rat and guinea-pig neurotens in receptors. Such species-related differences in neurotensin receptors should be considered when comparing the activity of neurotens in analogues in assays using tissue preparations from various animal species.
After the tridecapeptide neurotensln (NT) was isolated by Carraway and Leeman (I), the availability of NT synthetic partial sequences led to the observation by these authors that the C-termlnal hexapeptide (NT 8-13) was the minimal sequence required for the full expression of NT biological activity (2). This was later confirmed by several studies in which the activities of NT partial sequences and a number of NT analogues were measured in radloreceptor assays (3-5), in vivo (6,7) and in vitro (5,8-13) bloassays. Although most of these analogues displayed similar potencies in a variety of in vitro assays (5,11-13), there was one noticeable exception, the analogue [TrpI~]-NT in which a tryptophyl residue has been substituted for the tyrosyl residue at position II in the NT molecule (Table I). The biological activity of this analogue was tested in four assay systems, i.e. rat stomach strip, coronary vessels, portal vein, and gulnea-pig atria (11-13). In all the preparations derived from rat tissues, [TrplI]-NT was found to be slightly more active than NT (11-13). In contrast, [TrplI]-NT had only 3.2% of the activity of NT in isolated gulnea-plg atria (II). These observa-
Recipients of a fellowship from the D~l~gation G~n~rale ~ la Recherche Sclentiflque et Technique ( D ~ S T , Paris). To whom all correspondence and reprint requests should be addressed. 0024-3205/82/i i 1145-06503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
1146
Rat and Gulnea-plg Neurotensin Receptors
Vol. 31, No. Ii, 1982
tlons suggested to us that NT receptors in rat might be different from those in gulnea-plg with regard to thelr specificity towards NT and [Trpll]-NT. Furthermore this hypothesis is supported by a report (14) which compared the activity of xenopsin (XE) and NT in several bioassays. Xenopsln is an octapeptlde isolated from frog skin (15) whose C-terminal hexapeptlde amino acid sequence strongly resembles that of NT (Table I). In the positlon corresponding to Tyr II for NT, XE possesses a tryptophyl residue. It was found that XE and NT were approximately equipotent in in vltro bioassays with rat preparations (isolated stomach strip and duodenum). In contrast, XE was 5-10 times less potent than NT in assays with guinea-pig bioassays (isolated ileum and atrium) (14). The present study was designed to test the hypothesis that rat and gulnea~ pig receptors have a different specificity for NT congeners with a tryptophan substituted for Tyr II. NT, [TrplI]-NT and XE were compared for their ability to inhibit the binding of [SH]NT to rat and guinea-plg brain synaptic membranes, and for their effect on the contractile activity of rat and guinea-plg ileum.
TABLE I Amino-Acid Sequences of NT, [TrplI]-NT and XE Pep t id e
1
2
3
4
5
6
7
8
9
I0
ii
12
13
NT
pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-lle-Leu-OH
[ Trpl I ]-NT
pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Trp-lle-Leu-OH
XE
pGlu-Gly-Lys-Arg-Pro-Trp-lle-Leu-OH Materials and Methods
Peptides. The amlno-acid sequences of NT, [TrplI]-NT and XE are shown in Table I. Synthetic XE was purchased from Beckman Bioproducts Department (Geneva, Switzerland). NT and [TrplI]-NT were synthesized by three of us (C.L., C.G. and J.V.R.). The synthesis of NT has been previously reported (16). [TrplI]-NT was synthesized following the same general procedure as that described for NT. Details of this synthesis will be given elsewhere (manuscript in preparation). [3H]NT (specific activity 65 Ci/mmol) was prepared by Jean-Louis Morgat (Centre d'Etudes Nucl~aires de Saclay, Gif-sur-Yvette, France) as previously described (4). [3H]NT (specific activity 61 Ci/mmol) was also purchased from New England Nuclear (Paris, France). Radloreceptor assays. Assay conditions and properties of [3H]NT binding to rat brain synaptic membranes have been described elsewhere (4). Briefly,[3H]NT at 2 nM was incubated for 30 min at 24°C in 250 ~i of 50 mM Tris-HCl, pH 7.5, containing 0.2% bovine serLml albumin, 0.4 mg/ml membrane protein, and varying concentrations of unlabeled peptides. Bound peptide was separated from free peptide by filtration on Milllpore filters (EGWP 0.2 ~m). The specific binding was calculated as the difference between the amount of radioactivity bound in the absence and in the presence of an excess (i ~M) of unlabeled NT. The same assay conditions were used for measuring the binding of NT, [TrplI]-NT and XE to guineapig brain synaptic membranes. These membranes were prepared from whole guineapig brain (minus cerebellum) according to the method used for preparing rat brain membranes (4). Preliminary experiments indicated that the binding of [=HINT (2 nM) to guinea-pig brain membranes measured as a function of time, reached a plateau by 20 min (not shown). At the plateau, total binding represented about 1% of the radioactivity (30,000 cpm) present in the incubation medium and nonspecific binding amounted to 10% of total binding. These values are similar to those obtained with rat brain membranes (4).
Vol. 31, No. II, 1982
Rat and Guinea-pig Neurotensin Receptors
1147
Biological assays. NT has previously been shown to contract isolated longitudinal smooth muscle strips from the guinea-pig ileum (17,18). The contractions result from the stimulation by NT of intestinal cholinergic excitatory nerves, and are enhanced in the presence of the ant icholinesterase agent neostigmine (18). This has been the basis of a convenient bloassay for NT and related peptides (5,18). Briefly, longitudinal smooth muscle strips (3-4 cm in length) were dissected from the guinea-pig ileum and set up in a I0 ml organ bath for isometric tension recording. After equilibration of the preparations at 37°C in Tyrode solution, pH 7.4, containing 0.i ~M neosti~mine methylsulfate, non-cumulatlve concentratlon-response curves for NT, [Trp~I]-NT and XE were obtained as previously described (18). NT has been shown to relax the longitudinal muscle of rat ileal segments that were previously contracted by acetylcholine (19). In the present study, longitudinal smooth muscle strips from rat ileum were obtained using the procedure previously reported for guinea-plg preparations (18). The rat ileal strips (3-4 cm in length) were set up for tension recording in normal Tyrode solution. After equilibration of the preparations (60-90 min), concentration-response curves for the relaxing effect of NT, [TrpII]-NT and XE were obtained as follows : the muscle preparation was contracted with 0.5 ~M acetylcholine chloride and varying concentrations of a given peptide were added i min later. The preparation was washed I min after the addition of peptide and a new cycle was started 3-6 min after the washout. The peptide-induced relaxation was measured as the maximal drop in tension which occurred within 10-15 sec following the addition of peptide. During this 10-15 sec period, the fading of the contraction elicited by acetylcholine was negligible. Muscle preparations were allowed to rest for 15-20 min after a complete concentration-response curve was obtained. Results Concentratlon-response curves for the inhibition of [3HINT binding by NT, [Trp11] NT and XE, to rat and guinea-pig brain synaptic membranes are shown in Fig. i. The three peptides were virtually equipotent in the rat radioreceptor assay (Fig. I, left). By contrast, in the guinea-plg binding assay [Trp11]-NT and XE were much less potent than NT (Fig. i, right). Table II shows that the ICs0 value for NT was similar, about 4 nM, in both binding assays, whereas the ICs0 values for [Trp 11]-NT and XE were I0 times higher in the guinea-pig than in the rat radioreceptor assay. TABLE II Comparison of the Binding and Biological Activities of NT, Rat and Guinea-Pig Rat Peptide
NT [ Trpl I ] -NT XE
Binding assay ICs0 (nM) 4.0 4.3 4.0
Trp 11 -NT and XE in
Gulnea-pig Bioassay ECs0 (nM) 2.1 1.7 2.6
Binding assay ICs0 (nM) 3.7 40 46
Bioassay ECs0 (nM) 4.2 30 20
ICs0 (concentration of peptide that inhibits 50% of the binding of [3H]NT) and EC$0 (concentration of peptlde that produces half-maxlmal effect) were derived from Fig. 1 and 2, respectively. Concentratlon-response curves for the biological effect of NT, [TrpII]-NT and XE in rat and gulnea-plg ileum are shown in Fig. 2 (left) and Fig. 2 (right),
1148
Rat and Gulnea-plg Neurotensln Receptors
Vol. 31, No. 11, 1982
FIG. I Competitive inhibition by unlabeled NT (9~'Q), [Trp .I ]-NT (I~'~) and XE (A~4&) of [3H]NT binding to synaptic membranes from rat (left panel) and gulnea-plg (right panel) brain. Binding is expressed as the percentage of initial binding of [3H]NT, i.e. the binding of [3H]NT in the absence of unlabeled peptide. The non-speclfic binding has been subtracted. Points are the mean _+ SE from 7 and 3 experiments with unlabeled NT in the rat guinea-pig radioreceptor assays, respectively, and the mean from 3 and 2 experiments with [TrpII]-NT and XE in the rat and gulnea-plg binding assays, respectively. .
i i [m,~],~M
"~
-s
-7
-6
FIG. 2 Concentratlon-response curves for the effects of NT (9~'O), [TrpII]-NT ( m - l ) and XE (A--A) on the contractility of rat (left panel) and guineapig (right panel) ileum. Results are expressed as the percentage of the maximal effect induced by NT. Points are the mean _+ SE from 7, 4 and 4 experiments with NT, [Trpll]-NT and XE, respectively, in the rat assay, and from 7, 3 and 4 experiments with NT, 'ITrpII]-NT and XE, respectively, in the guinea-plg assay.
Vol. 31, No. ii, 1982
Rat and Guinea-pig Neurotensin Receptors
1149
respectively. The three peptides exhibited similar potency in the rat bioassay. In the guinea-pig bioassay, the ~otencies of [TrplI]-NT and XE were lower than that of NT. In both assays, [Trp I]-NT and XE were as effective as NT, indicating that these peptides behaved llke full agonists. ECs0 values derived from the curves in Fig. 2 are shown in Table II. The values were close to 2 nM for all three pe~tides in the rat bioassay. In the guinea-pig bloassay, the ECs0 values for [Trp I]-NT and XE were 5-7 times higher than that for NT (4.2 nM). The relative potency of [TrplI]-NT in the binding and biological assays described here are shown in Table III together with the potency values obtained by others in various i__~nvitro assays (13). Comparison among the values derived from 5 rat and 3 guinea-pig assay systems involving brain tissues as well as peripheral tissues such as gastro-intestlnal or vascular smooth muscle, and cardiac muscle shows that [TrplI]-NT had 93-130% of the activity of NT in all assays with rat preparations, and only 3-14% in assays with guinea-pig preparations. TABLE III Relative P~tency of ~Trp II]-NT in Binding and Biological Assays from Rat and Guinea-Pig-. Rat Synaptic Membranes
lleum
93
124
Stomach Strip 13~
Guinea-plg Coronary Vessels I0~
Portal Vein I15b
Synaptic Membranes 9.2
lleum
14
Atria
3.2 b
In all assays, potency values are expressed relative to NT taken as I00. Values derived from reference (13). Discussion The data presented here have clearly shown that, in the gulnea-pig, [Trp II ]-NT and XE are less active than NT. However, in the rat, the three peptldes are nearly equipotent in binding to NT receptors and in activating these receptors. Our results are in agreement with those of others (11-13) who have measured the activity of [Trp II ]-NT in several bloassays using rat and guinea-pig tissues (Table III). The present results also agree with the report (14) that XE was almost as potent as NT in the rat, whereas XE was 5-10 times less potent than NT in the gulnea-pig. Taken together, these observations show that, unlike NT receptors in rat, the receptors in guinea-plg discriminate between NT and NT analogues in which a tryptophan replaces the tyroslne normally present at position II of the NT molecule. This strongly suggests that NT receptors in the rat differ from those in the guinea-pig. However, it should be noted that a n~nber of NT partial sequences and analogues, for example [Phe II ]-NT, were shown to have similar potencies in various rat and gulnea-plg assays (5, 8-13). Therefore, it appears that rat and guinea-pig NT receptors are not grossly different but rather exhibit limited differences which can be detected with NT analogues bearing appropriate modifications. Whether such differences can be observed in other species and with other NT analogs is not known at present. However, this possibility should be considered when comparing the activity of NT analogues in bloassays and binding assays using tissue preparations from various animal species. Previous studies with NT analogues modified at position II of the HT amino acid sequence have revealed that Tyr 1 ! plays a crucial role in the binding of NT to its receptors (5) and for the expression of biological activity (5-7, 10-13). Furthermore, modifications of position II have led to two NT analogues ([D-Trp II ]NT and [Tyr(Me~ I ]-NT) which behave as partial NT antagonists in certain tissue preparations and as weak agonlsts in other preparations, thus suggesting some
1150
Rat and Gulnea-plg Neurotensln Receptors
Vol. 31, No. ii, 1982
degree of tissue-related differences among NT receptors (13), Finally, comparisons of the in vivo and in vitro activities of NT analogues with D-amlno acids at position II have suggested that Tyr II is involved in the inactivation of NT in brain tissues (5). These observations, together with the present study, suggest that appropriate modifications or substitutions at the key position II of the NT molecule may provide NT analogues with interesting properties such as NT antagonists, analogues resistant to degradation, and species- or tissuespecific analogues. Acknow led~ement s We thank Jean-Louls Morgat (Centre d'Etudes Nucl~alres de Saclay, Gif-sur-Yvette, France) for the gift of [3H]NT, Ellen Van Obberghen-Schilling for careful reading of the manuscript, and Catherine Roulinat for expert secretarial assistance. This work was supported in part by grant 81.E.0542 from the D~l~gation G~n~rale la Recherche Sclentifique et Technique (DGRST, Paris) and by the Fondation pour la Recherche M~dicale. References i. 2.
3.
4.
5.
6. 7. 8. 9. I0. II. 12. 13. 14. 15. 16. 17. 18. 19.
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