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Comparison of some biological properties of neurotensin and its natural analogue LANT-6
European Journal of Pharmacology, 99 (1984) 357-360
357
Elsevier Short communication
C O M P A R I S O N O F S O M E B I O L O G I C A L P R O P E R T I E S OF N E U R O T E N S I N AND I T S N A T U R A L A N A L O G U E LANT-6 PATRICK KITABGI *, FRI~DI~RICCHECLER and JEAN-PIERRE VINCENT Centre de Biochimie du Centre National de la Recherche Seientifique (CNRS), Facultb des Sciences, Parc Valrose, 06034 Nice Cedex, France
Received 30 January 1984, accepted 7 February 1984
P. KITABGI, F. CHECLER and J.P. VINCENT, Comparison of some biological properties of neurotensin and its natural analogue L A N T - 6 , European J. Pharmacol. 99 (1984) 357-360. The biological properties of neurotensin (NT) and the chicken peptide [Lys8,Asng]neurotensin-(8-13) (LANT-6) were compared in 3 in vitro assays: (1) competitive inhibition of [3HINT binding to rat brain synaptic membranes, (2) relaxation of the rat ileum, and (3) contraction of the guinea-pig ileum. The data show that LANT-6 interacted with NT receptors like a weak agonist having full intrinsic activity and low affinity. LANT-6 is unlikely to represent a physiological activator of NT receptors. Neurotensin-related peptides
Radioreceptor assay
1. Introduction
Recently a new peptide from the neurotensin (NT) family [Lys8,Asn9]NT-(8-13) (LANT-6), was purified and characterized from chicken small intestine (Carraway and Ferris, 1983). Using radioimmunoassay techniques, this peptide was found to be widely distributed throughout chicken brain and gastrointestinal tract (Carraway et al., 1983). In addition, immunoreactive LANT-6 was shown to be present in extracts of rat brain and small intestine (Carraway et al., 1983). With respect to its biological properties, LANT6 was found to produce effects qualitatively similar to those of N T when injected intravenously into anesthetized rats, i.e. a pressor-depressor effect on blood pressure (although the pressor response was relatively higher with LANT-6 than with NT), an increase in blood glucose, cyanosis and increase in hematocrit (Carraway and Ferris, 1983). However, the doses of LANT-6 required for these effects were about two orders of magnitude
* To whom all correspondence should be addressed. 0014-2999/84/$03.00 © 1984 Elsevier Science Publishers B.V.
Bioassay
Rat ileum
Guinea-pig ileum
higher than similarly effective doses of N T (Carraway and Ferris, 1983). Since the biological activities of LANT-6 and N T were compared using only peripheral in vivo bioassays, we thought it might be interesting to test the in vitro activity of both peptides using central and peripheral bioassay systems. In the present study, LANT-6 and N T were compared for their ability to inhibit the binding of [3H]NT to rat brain synaptic membranes and for their effect on the contractile activity of rat and guineapig ileum preparations in vitro. 2. Materials and methods 2.1. Peptides
Native LANT-6 was a generous gift of R. Carraway (University of Massachusetts Medical School, Worcester, MA, USA). Synthetic N T was provided by C. Granier and J. Van Rietschoten (Facult6 de Mrdecine Nord, Marseille, France). [3H]NT (specific radioactivity 61 C i / m m o l ) was purchased from New England Nuclear (Paris, France).
358
2.2. Binding and biologic assays Assay conditions and properties of [3H]NT binding to rat brain synaptic membranes have been described elsewhere (Kitabgi et al., 1977). The ability of unlabelled N T and LANT-6 to inhibit the specific binding of [3H]NT to rat brain synaptic membranes was measured as described previously for a variety of N T partial sequences and analogues (Kitabgi et al., 1980). N T has been shown to contract isolated longitudinal smooth muscle strips from guinea-pig ileum through a nerve-mediated, cholinergic process, an effect which is potentiated by the anticholinesterase agent neostigmin Kitabgi and Freychet, 1979). This bioassay yielded concentration-response curves for N T and L A N T - 6 as reported previously for NT-related peptides (Kitabgi et al., 1980). The property of N T to induce myogenic relaxation of rat ileal longitudinal smooth muscle strips precontracted by acetylcholine has been used previously as a bioassay for N T and related peptides (Checler et al., 1982). This same bioassay was used here to compare the biological activity of LANT-6 and NT.
3. Results
Like NT, LANT-6 exerted a concentration-dependent inhibition of [3H]NT specific binding to rat brain synaptic membranes as shown in fig. 1A. At a sufficiently high concentration, the peptide completely inhibited the specific binding of [3 H]NT to its brain receptors. LANT-6 also elicited concentration-dependent relaxation of rat ileum (fig. 1B) and contraction of guinea-pig ileum (fig. 1C). The contractile responses of these tissues to LANT-6 were qualitatively indistinguishable from the responses to NT, i.e. LANT-6 did not produce any detectable effect on muscle tension that was not observed with N T (not shown). Furthermore, LANT-6 elicited the same maximal response as N T in both rat and guinea-pig ileum preparations (fig. 1B and C). However, comparison of the concentration-response curves for N T and LANT-6 in fig. 1, shows that LANT-6 was much less potent than N T in all
o .4/
-9
-8
-7
-6
-5
,
_,
.,
.,
_,
B
Fig. 1. (A) Competitive inhibition of [3H]NT binding to rat brain synaptic membranes by increasing concentrations of N T (o o, 7 experiments) and LANT-6 ( i i , 2 experiments); (B) Concentration-response curves for the relaxation of the rat ileum by N T (O O, 7 experiments) and LANT-6 (--II, 3 experiments); (C) Concentration-response curves for the contraction of guinea-pig ileum by N T (O O, 7 experiments) and LANT-6 ( i i , 4 experiments).
TABLE 1 Comparison of the binding and biological activities in vitro of NT, LANT-6, and NT-(8-13). Peptide
Binding to rat Relaxation of Contraction of brain membranes rat ileum guinea-pig ileum ICso, nM a ECso, n M a ECs0, nM a
NT 3.5 LANT-6 1780 (0.2) b NT-(8-13) (15.0) b,¢
2.1 180 (1.2) b
4.2 210 (2.0) b (20.0) b,~
a iC5o (concentration of peptide that inhibited 50% of the binding of [3HINT) and ECso (concentration of peptide that elicited half maximal effect) were derived from fig. 1. b The values in parentheses represent the potency of LANT-6 and NT-(8-13) relative to that of N T taken as 100 in each assay system. Potency values for NT-(8-13) were taken from Kitabgi et al. (!980) and Granier et al. (1982).
359
three assay systems. The IC50 and ECs0 values for LANT-6 and NT, as well as the potency of LANT-6 relative to that of N T in the three bioassays are shown in table 1. On a molar basis LANT-6 was 500 times less potent than N T in binding to rat brain N T receptors, and about 50-100 less potent than N T in affecting the contractile activity of rat and guinea-pig ileum. Because LANT-6 is an analogue of the C-terminal hexapeptide sequence of NT, i.e. NT-(8-13), potency values for synthetic NT-(8-13), as derived from previous studies (Kitabgi et al., 1980; Granier et al., 1982) were included in table 1. Although NT-(8-13) displayed a 5-6 fold reduction in activity as compared to NT, it was still 10-70 times more potent than LANT-6.
4. Discussion The present data show that in three in vitro bioassay systems designed to assess the interaction of peptides with central and peripheral N T receptors, the chicken N T variant LANT-6 displayed the same intrinsic activity as N T although its affinity was much lower than that of the parent peptide. LANT-6 can therefore be defined as a N T receptor agonist of low affinity and full intrinsic activity. LANT-6 (H-Lys-Asn-Pro-Tyr-Ile-Leu-OH) resembles the C-terminal hexapeptide of N T (HArg-Arg-Pro-Tyr-Ile-Leu-OH), except that the chicken peptide has a lysine and an asparagine residue substituted for the N T residues Arg 8 and Arg 9, respectively, hence the designation [LysS,Asn9]NT-(8-13) abbreviated LANT-6 (Carraway and Ferris, 1983). It is generally recognized that the C-terminal hexapeptide of NT, NT-(8-13), contains all the structural requirements for binding to N T receptors and expression of biological activity (see Kitabgi, 1982). It has been shown previously that the side-chain positive charges of N T residues 8 and 9 play an important role in the interaction of N T with its receptors: substitution of either Arg 8 or Arg 9 in NT-(8-13), by a lysine residue does not modify the peptide activity, whereas substitution by uncharged residues like citrulline or acetyl-lysine brings about a 50-100
fold decrease in biological activity (Kitabgi et al., 1980; Granier et al., 1982). It would be expected therefore that substitution of Arg 9 by an asparagine residue, as in LANT-6, results in a similar decrease in biological activity and, indeed, this was observed in the present study. It should be noted that the low in vitro potency of LANT-6 in eliciting NT-like effects, as documented here, agrees well with its reported low in vivo potency (Carraway and Ferris, 1983). Together, these observations suggest that LANT-6 is unlikely to represent a physiological activator of N T receptors. The question has been raised whether LANT-6 possesses its own spectrum of biological effects mediated via its own set of receptors (Carraway and Ferris, 1983). Although the present data show that LANT-6 interacts with NT-preferring receptors (receptors with high affinity for N T and low affinity for LANT-6), they provide no evidence for the existence of LANT-6-preferring receptors. Such receptors, if they exist would be expected to exhibit high affinity for LANT-6 and either no affinity or more likely low affinity for N T in view of the structural homology between the two peptides. Binding experiments using avian and mammalian brain and gut tissues and radiolabelled LANT-6 should prove essential in searching for LANT-6 receptors.
Acknowledgements We wish to thank G. C16net for expert secretarial assistance.
References Carraway, R.E. and C.F. Ferris, 1983, Isolation, biological and chemical characterization, and synthesis of a neurotensinrelated hexapeptide from chicken intestine, J. Biol. Chem. 258, 2475. Carraway, R.E., S.E. Ruane and R.S.Ritsema, 1983, Radioimmunoassay for Lys 8, Asn9, Neurotensin 8-13: Tissue and subcellular distribution of immunoreactivity in chickens, Peptides 4, 111. Checler, F., C. Labb~, C. Granier, J. Van Rietschoten, P. Kitabgi and J.P. Vincent, 1982, [Trp11]-neurotensin and xenopsin discriminate between rat and guinea-pig neurotensin receptors, Life Sci. 31, 1145.
360 Granier, C., J. van Rietschoten, P. Kitabgi, C. Poustis and P. Freychet, 1982, Synthesis and characterization of neurotensin analogues for structure/activity relationship studies. Acetyl-neurotensin-(8-13) is the shortest analogue with full binding and pharmacological activities, European J. Biochem. 124, 117. Kitabgi, P., 1982, Effects of neurotensin on intestinal smooth muscle: application to the study of structure-activity relationships, Ann. N.Y. Acad. Sci. 400, 37. Kitabgi, P., R. Carraway, J. Van Rietschoten, C. Granier, J.L. Morgat, A. Menez, S. Leeman and P. Freychet, 1977,
Neurotensin: Specific binding to synaptic membranes from rat brain, Proc. Natl. Acad. Sci. U.S.A. 74, 1846. Kitabgi, P. and P. Freychet, 1979, Neurotensin contracts the guinea-pig longitudinal ileal smooth muscle by inducing acetylcholine release, European J. Pharmacol. 56, 403. Kitabgi, P., C. Poustis, C. Granier, J. van Rietschoten, J. Rivier, J.L. Morgat and P. Freychet, 1980, Neurotensin binding to extraneural and neural receptors: comparison with biological activity and structure-activity relationships, Mol. Pharmacol. 18, 11.
357
Elsevier Short communication
C O M P A R I S O N O F S O M E B I O L O G I C A L P R O P E R T I E S OF N E U R O T E N S I N AND I T S N A T U R A L A N A L O G U E LANT-6 PATRICK KITABGI *, FRI~DI~RICCHECLER and JEAN-PIERRE VINCENT Centre de Biochimie du Centre National de la Recherche Seientifique (CNRS), Facultb des Sciences, Parc Valrose, 06034 Nice Cedex, France
Received 30 January 1984, accepted 7 February 1984
P. KITABGI, F. CHECLER and J.P. VINCENT, Comparison of some biological properties of neurotensin and its natural analogue L A N T - 6 , European J. Pharmacol. 99 (1984) 357-360. The biological properties of neurotensin (NT) and the chicken peptide [Lys8,Asng]neurotensin-(8-13) (LANT-6) were compared in 3 in vitro assays: (1) competitive inhibition of [3HINT binding to rat brain synaptic membranes, (2) relaxation of the rat ileum, and (3) contraction of the guinea-pig ileum. The data show that LANT-6 interacted with NT receptors like a weak agonist having full intrinsic activity and low affinity. LANT-6 is unlikely to represent a physiological activator of NT receptors. Neurotensin-related peptides
Radioreceptor assay
1. Introduction
Recently a new peptide from the neurotensin (NT) family [Lys8,Asn9]NT-(8-13) (LANT-6), was purified and characterized from chicken small intestine (Carraway and Ferris, 1983). Using radioimmunoassay techniques, this peptide was found to be widely distributed throughout chicken brain and gastrointestinal tract (Carraway et al., 1983). In addition, immunoreactive LANT-6 was shown to be present in extracts of rat brain and small intestine (Carraway et al., 1983). With respect to its biological properties, LANT6 was found to produce effects qualitatively similar to those of N T when injected intravenously into anesthetized rats, i.e. a pressor-depressor effect on blood pressure (although the pressor response was relatively higher with LANT-6 than with NT), an increase in blood glucose, cyanosis and increase in hematocrit (Carraway and Ferris, 1983). However, the doses of LANT-6 required for these effects were about two orders of magnitude
* To whom all correspondence should be addressed. 0014-2999/84/$03.00 © 1984 Elsevier Science Publishers B.V.
Bioassay
Rat ileum
Guinea-pig ileum
higher than similarly effective doses of N T (Carraway and Ferris, 1983). Since the biological activities of LANT-6 and N T were compared using only peripheral in vivo bioassays, we thought it might be interesting to test the in vitro activity of both peptides using central and peripheral bioassay systems. In the present study, LANT-6 and N T were compared for their ability to inhibit the binding of [3H]NT to rat brain synaptic membranes and for their effect on the contractile activity of rat and guineapig ileum preparations in vitro. 2. Materials and methods 2.1. Peptides
Native LANT-6 was a generous gift of R. Carraway (University of Massachusetts Medical School, Worcester, MA, USA). Synthetic N T was provided by C. Granier and J. Van Rietschoten (Facult6 de Mrdecine Nord, Marseille, France). [3H]NT (specific radioactivity 61 C i / m m o l ) was purchased from New England Nuclear (Paris, France).
358
2.2. Binding and biologic assays Assay conditions and properties of [3H]NT binding to rat brain synaptic membranes have been described elsewhere (Kitabgi et al., 1977). The ability of unlabelled N T and LANT-6 to inhibit the specific binding of [3H]NT to rat brain synaptic membranes was measured as described previously for a variety of N T partial sequences and analogues (Kitabgi et al., 1980). N T has been shown to contract isolated longitudinal smooth muscle strips from guinea-pig ileum through a nerve-mediated, cholinergic process, an effect which is potentiated by the anticholinesterase agent neostigmin Kitabgi and Freychet, 1979). This bioassay yielded concentration-response curves for N T and L A N T - 6 as reported previously for NT-related peptides (Kitabgi et al., 1980). The property of N T to induce myogenic relaxation of rat ileal longitudinal smooth muscle strips precontracted by acetylcholine has been used previously as a bioassay for N T and related peptides (Checler et al., 1982). This same bioassay was used here to compare the biological activity of LANT-6 and NT.
3. Results
Like NT, LANT-6 exerted a concentration-dependent inhibition of [3H]NT specific binding to rat brain synaptic membranes as shown in fig. 1A. At a sufficiently high concentration, the peptide completely inhibited the specific binding of [3 H]NT to its brain receptors. LANT-6 also elicited concentration-dependent relaxation of rat ileum (fig. 1B) and contraction of guinea-pig ileum (fig. 1C). The contractile responses of these tissues to LANT-6 were qualitatively indistinguishable from the responses to NT, i.e. LANT-6 did not produce any detectable effect on muscle tension that was not observed with N T (not shown). Furthermore, LANT-6 elicited the same maximal response as N T in both rat and guinea-pig ileum preparations (fig. 1B and C). However, comparison of the concentration-response curves for N T and LANT-6 in fig. 1, shows that LANT-6 was much less potent than N T in all
o .4/
-9
-8
-7
-6
-5
,
_,
.,
.,
_,
B
Fig. 1. (A) Competitive inhibition of [3H]NT binding to rat brain synaptic membranes by increasing concentrations of N T (o o, 7 experiments) and LANT-6 ( i i , 2 experiments); (B) Concentration-response curves for the relaxation of the rat ileum by N T (O O, 7 experiments) and LANT-6 (--II, 3 experiments); (C) Concentration-response curves for the contraction of guinea-pig ileum by N T (O O, 7 experiments) and LANT-6 ( i i , 4 experiments).
TABLE 1 Comparison of the binding and biological activities in vitro of NT, LANT-6, and NT-(8-13). Peptide
Binding to rat Relaxation of Contraction of brain membranes rat ileum guinea-pig ileum ICso, nM a ECso, n M a ECs0, nM a
NT 3.5 LANT-6 1780 (0.2) b NT-(8-13) (15.0) b,¢
2.1 180 (1.2) b
4.2 210 (2.0) b (20.0) b,~
a iC5o (concentration of peptide that inhibited 50% of the binding of [3HINT) and ECso (concentration of peptide that elicited half maximal effect) were derived from fig. 1. b The values in parentheses represent the potency of LANT-6 and NT-(8-13) relative to that of N T taken as 100 in each assay system. Potency values for NT-(8-13) were taken from Kitabgi et al. (!980) and Granier et al. (1982).
359
three assay systems. The IC50 and ECs0 values for LANT-6 and NT, as well as the potency of LANT-6 relative to that of N T in the three bioassays are shown in table 1. On a molar basis LANT-6 was 500 times less potent than N T in binding to rat brain N T receptors, and about 50-100 less potent than N T in affecting the contractile activity of rat and guinea-pig ileum. Because LANT-6 is an analogue of the C-terminal hexapeptide sequence of NT, i.e. NT-(8-13), potency values for synthetic NT-(8-13), as derived from previous studies (Kitabgi et al., 1980; Granier et al., 1982) were included in table 1. Although NT-(8-13) displayed a 5-6 fold reduction in activity as compared to NT, it was still 10-70 times more potent than LANT-6.
4. Discussion The present data show that in three in vitro bioassay systems designed to assess the interaction of peptides with central and peripheral N T receptors, the chicken N T variant LANT-6 displayed the same intrinsic activity as N T although its affinity was much lower than that of the parent peptide. LANT-6 can therefore be defined as a N T receptor agonist of low affinity and full intrinsic activity. LANT-6 (H-Lys-Asn-Pro-Tyr-Ile-Leu-OH) resembles the C-terminal hexapeptide of N T (HArg-Arg-Pro-Tyr-Ile-Leu-OH), except that the chicken peptide has a lysine and an asparagine residue substituted for the N T residues Arg 8 and Arg 9, respectively, hence the designation [LysS,Asn9]NT-(8-13) abbreviated LANT-6 (Carraway and Ferris, 1983). It is generally recognized that the C-terminal hexapeptide of NT, NT-(8-13), contains all the structural requirements for binding to N T receptors and expression of biological activity (see Kitabgi, 1982). It has been shown previously that the side-chain positive charges of N T residues 8 and 9 play an important role in the interaction of N T with its receptors: substitution of either Arg 8 or Arg 9 in NT-(8-13), by a lysine residue does not modify the peptide activity, whereas substitution by uncharged residues like citrulline or acetyl-lysine brings about a 50-100
fold decrease in biological activity (Kitabgi et al., 1980; Granier et al., 1982). It would be expected therefore that substitution of Arg 9 by an asparagine residue, as in LANT-6, results in a similar decrease in biological activity and, indeed, this was observed in the present study. It should be noted that the low in vitro potency of LANT-6 in eliciting NT-like effects, as documented here, agrees well with its reported low in vivo potency (Carraway and Ferris, 1983). Together, these observations suggest that LANT-6 is unlikely to represent a physiological activator of N T receptors. The question has been raised whether LANT-6 possesses its own spectrum of biological effects mediated via its own set of receptors (Carraway and Ferris, 1983). Although the present data show that LANT-6 interacts with NT-preferring receptors (receptors with high affinity for N T and low affinity for LANT-6), they provide no evidence for the existence of LANT-6-preferring receptors. Such receptors, if they exist would be expected to exhibit high affinity for LANT-6 and either no affinity or more likely low affinity for N T in view of the structural homology between the two peptides. Binding experiments using avian and mammalian brain and gut tissues and radiolabelled LANT-6 should prove essential in searching for LANT-6 receptors.
Acknowledgements We wish to thank G. C16net for expert secretarial assistance.
References Carraway, R.E. and C.F. Ferris, 1983, Isolation, biological and chemical characterization, and synthesis of a neurotensinrelated hexapeptide from chicken intestine, J. Biol. Chem. 258, 2475. Carraway, R.E., S.E. Ruane and R.S.Ritsema, 1983, Radioimmunoassay for Lys 8, Asn9, Neurotensin 8-13: Tissue and subcellular distribution of immunoreactivity in chickens, Peptides 4, 111. Checler, F., C. Labb~, C. Granier, J. Van Rietschoten, P. Kitabgi and J.P. Vincent, 1982, [Trp11]-neurotensin and xenopsin discriminate between rat and guinea-pig neurotensin receptors, Life Sci. 31, 1145.
360 Granier, C., J. van Rietschoten, P. Kitabgi, C. Poustis and P. Freychet, 1982, Synthesis and characterization of neurotensin analogues for structure/activity relationship studies. Acetyl-neurotensin-(8-13) is the shortest analogue with full binding and pharmacological activities, European J. Biochem. 124, 117. Kitabgi, P., 1982, Effects of neurotensin on intestinal smooth muscle: application to the study of structure-activity relationships, Ann. N.Y. Acad. Sci. 400, 37. Kitabgi, P., R. Carraway, J. Van Rietschoten, C. Granier, J.L. Morgat, A. Menez, S. Leeman and P. Freychet, 1977,
Neurotensin: Specific binding to synaptic membranes from rat brain, Proc. Natl. Acad. Sci. U.S.A. 74, 1846. Kitabgi, P. and P. Freychet, 1979, Neurotensin contracts the guinea-pig longitudinal ileal smooth muscle by inducing acetylcholine release, European J. Pharmacol. 56, 403. Kitabgi, P., C. Poustis, C. Granier, J. van Rietschoten, J. Rivier, J.L. Morgat and P. Freychet, 1980, Neurotensin binding to extraneural and neural receptors: comparison with biological activity and structure-activity relationships, Mol. Pharmacol. 18, 11.