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Evidence for biological activity of two N-terminal fragments of neurotensin, neurotensin1–8 and neurotensin1–10
Brain Research, 301 (1984) 153-156 Elsevier
153
BRE 20213
Evidence for biological activity of two N-terminal fragments of neurotensin, neurotensinl_8 and neurotensinl_10 DANIEL E. HERNANDEZ I, CHARLES M. RICHARDSON l, CHARLES B. NEMEROFF5, ROY C. ORLANDO-', SERGE ST-PIERRE6, FRANCIS RIOUX~and ARTHUR J. PRANGE, Jr. 1.3.4
1Biological Sciences Research Center, Departments ofeMedicine and 3Psychiatry and 4The Neurobiology Program, University of North Carolina School of Medicine, Chapel Hill. NC, 5 Department of Psychiatry, Duke University, Durham, NC ( U. S. A. ) and 6Departments of Physiology and Pharmacology, University of Sherbrooke, Sherbrooke, Que. (Canada) (Accepted January 31st, 1984)
Key words: neurotensin- - neurotensin-fragments - - gastric - - cytoprotection
Intracisternal (i.c.) administration of the endogenous tridecapeptide neurotensin (NT) has been previously shown to significantly reduce the incidence of cold-restraint stress (CRS)-induced gastric ulcers in rats. In this study we confirm the cytoprotective activity of central NT, and document structure-activity relationships for this effect of NT. When tested in a dose equimolar to 17.9 nmol of NT the NT analogs [GIn4]NT, D-TrpII-NT, and D-ArgS-NTwere cytoprotective, whereas o-Argg-NT was not. Gonadotropin-releasing hormone (Gn-RH) and melanocyte-stimulating hormone-release inhibiting factor (MIF-1), two oligopeptides structurally unrelated to NT exhibited no cytoprotective efficacy in this paradigm. The C-terminal fragments of NT, xenopsin and NT8_13, and the N-terminal fragment NTI_6 were completely ineffective. Finally, NT I ~ and NTH0, two N-terminal fragments of NT produced significant cytoprotective activity at this dose level. The cytoprotection afforded by NTI~ and NTH0, like that of NT, was dose-dependent with EDs0's similar to that of NT (NT = 16.2 nmol, NTt_s = 17.8 nmol and NT~ l0 = 19.9 nmol). In conclusion, we demonstrate that smaller molecular weight forms of NT thought to be degradation products of NT can effectively exert biological effects.
Neurotensin (NT), a tridecapeptide ( p G l u - L e u Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-lle-LeuOH), was first isolated from bovine hypothalami 3. Immunohistochemical and radioimmunological evidence has revealed a widespread distribution of NT in the central nervous system (CNS) and in n o n - n e u ral peripheral tissues, including the gastrointestinal tract, of several vertebrate species 4. In the CNS, N T has been found in the synaptosomal fraction after density gradient centrifugation, and specific high-affinity receptors for NT have been identified in brain m e m b r a n e s 12. Further, depolarizing stimuli have been shown to produce a calciumdependent release of immunoreactive NT from brain tissue slices, suggesting that N T may serve a role in neurotransmission ~0. Consistent with this hypothesis is the considerable body of evidence indicating that
brain NT may play a physiological role in thermoregulation 2.s, antinociception 16, and m a i n t e n a n c e of gastric mucosal integrity iT. These effects of NT appear to be mediated within the CNS, because peripheral administration of NT is devoid of either hypothermic, antinociceptive or cytoprotective activity9A7. However, NT-induced cytoprotection has also been observed after peripheral injection in the restraintwater immersion test 20. Degradation of N T by peptidases has been demonstrated in brain as well as in peripheral plasmal,5, 7A5. After in vitro incubation in a rat brain m e m b r a n e preparation, the main product in both soluble and particulate fractions are NTI_8 and the C-terminal fragment NTg_13. In addition, the Nterminal fragment NTI_8 appears to be relatively resistant to further degradation, whereas NT9_13, in contrast, is rapidly degradated to liberate tyrosine as
Correspondence: D. E. Hernandez, Biological Sciences Research Center 220-H, University of North Carolina School of Medicine, Chapel Hill, NC 27514, U.S.A. 0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V.
154 a result of a post-proline cleavage at Pro l0 (ref. 15). The decapeptide N-terminal fragment (NTH0) has also been identified after in vivo incubation, but proportionally more of this decapeptide can be identified from the particulate, rather than the soluble fraction'S. The results of incubation of NT in rat plasma have also been studied in some detail. Intravenous injection of radiolabeled [3H]NT has been shown to generate NTI_8, NTl_ll and NT9_13. The apparent halflives of intravenously injected synthetic NTI_s and NT1_I1 are 9.5 and 5 min, respectively, while that of NTg-13 is less than 0.5 min~. In this report we demonstrate that two N-terminal fragments of NT, NTI_s and NTl_10, exert significant cytoprotective activity as measured by their ability to significantly prevent the development of cold-restraint-stress (CRS)-induced gastric ulcers in rats after intracisternal (i.c.) injection. Adult male Sprague-Dawley rats (220-250 g) from Zivic-Miller (Cincinnati, OH) were kept in an environmentally controlled room on a 12 h dark: 12 h light schedule (lights on 06.00 h), and fed laboratory chow and water ad libitum. They were maintained in the animal facility for at least one week prior to experimental use. All rats were deprived of food but not water 24 h prior to the experiments. Peptides or vehicle (10 ¢tl of 0.9% NaCI) were administered i.c. under light ether anesthesia as described elsewhere 17. Immediately after the i.c. injection animals were restrained in wire mesh and placed supine in a cold (4 °C) room for 3 h. This regimen of physical restraint and simultaneous exposure to a cold environment has been reported previously to reliably produce ulcers in the glandular portion of the stomach of food-deprived rats 9.17. Following the 3 h stress period, rats were killed by decapitation and the stomach examined for gastric ulcerations by a trained observer with no knowledge of the treatment regimen. The presence of gastric ulcers was assessed by using the following scale: 0, no pathology; 1+ mucosal edema and/or petechiae; and 2+ gross gastric mucosal ulceration. The results, which are expressed as cytoprotective response, represent the percentage of rats that exhibited no gastric lesions. In the first series of experiments all substances were initially administered i.c. in a dose equimolar to 17.9 nmol of NT. This dose of NT was chosen be-
cause it has been previously demonstrated to produce a significant cytoprotective response in this model 9.17. The cytoprotective response of each treatment group was compared to that of the saline-treated controls. Statistical significance was assessed with the Kruskal-Wailis test for multiple comparisons of non-parametric data. Because the two NT fragments, NT~_8 and NTI_I0, elicited an almost identical cytoprotective effect when tested at a single dose level as did NT itself, dose-response studies were performed by administering different doses of NT and these two NT fragments to allow direct comparison with the parent compound (NT). The dose-response curves were generated with the least-square method, followed by simple linear regression analysis; P values for the curves were determined with the Chi-square test. In addition, the EDso for NT and both NT fragments was determined from the dose-response curves using the probit procedure. In all the statistical tests, P values of 0.05 or less were considered to represent significant differences between treatment groups. Neurotensin, xenopsin, gonadotropin-releasing hormone (Gn-RH) and melanocyte-stimulating hormone-release-inhibiting-factor (MIF-I) were obtained from Bachem (Torrance, CA). Two NT fragments, NT~_6 and NTI_~, were purchased from Cambridge Research Biochemicals Ltd. (Harston, Cambridgeshire, U.K.), and [Gin4]neurotensin from Peninsula Laboratories (San Carlos, CA). The following structural analogues of NT: DArgS-NT, D-Arg9-NT, D-Trp I t-NT and the NT fragments, NTs_E3and NT 1 l/~, were synthetized by one of us (S.St-P), using solid phase methodology as previously described TM. The results obtained in the first set of experiments, which are presented in Table I, show that, as previously demonstratedg,l 7. i.c. NT (17.9 nmol) produced significant gastric cytoprotection. When tested in a dose equimolar to 17.9 nmol of NT, the N-terminal fragment NTI_6 as well as the C-terminal fragment NTs-13 were not cytoprotective. Furthermore, xenopsin, an octapeptide which closely resembles the C-terminal fragment of NT, and therefore can be considered a natural analogue of NT8-13, was also ineffective. The peptides structurally unrelated to NT including GnRH and M1F-I exhibited no cytoprotective efficacy in this test.
155 TABLE I
Effects of centrally administered peptides on cold-restraint stress (CRS)-induced gastric ulcers in rats All substances were administered intracisternally in a dose equimolar to 17.9 nmol of neurotensin.
Peptide
n
Cytoprotective response
(%) Saline Neurotensin [GIn4]NT o-Trpll-NT D-ArgS-NT NTI_10 NTI~s D-Argg-NT Xenopsin NT8_13 NTv, MIF-I Gn-RH
60 40 10 10 10 13 12 10 9 10 10 10 10
32.3 85* 100"* 100"* 90* 86.7* 75* 60 55.6 50 50. 50 40
** P < 0.001, * P < 0.01 Kruskal-Wallis test (chi-square approximation). All comparisons were done against saline-treated controls.
The fact that NTv.6 and the C-terminal fragments of NT (NT8_13, xenopsin) are ineffective, suggsts the hypothesis that the middle portion of the NT molecule (-Pro7-ArgS-Arg9-Pro 10) appears to play a critical role for the expression of biological activity in this test. Both NT~_8 and NTI_~0 exhibited significant and substantial cytoprotective efficacy at this dose level (Table I). We next examined this hypothesis by testing several NT analogues with single stereoisomeric D-amino acid replacements for the naturally occurring Lforms. The fact that D-ArgS-NT, but not D-Arg9-NT produced a significant cytoprotective response similar to NT, indicates that within this critical region there appears to be some degree of conformational specificity. These results are concordant with findings pertaining to hypothermi# 4. From structure-activity studies it has been reported that D-Arg9-NT, and the C-terminal fragment NT9._13, but not DArgS-NT, are devoid of hypothermic activity. Furthermore, systematic substitution of the arginine residues at position 8 and 9 in NT by L-Ala have revealed that both arginine residues are required for optimal recognition by mast cell receptors, and it has been proposed that Arg 8 plays a greater role than Arg 9 in this particular radioreceptor assay 13. This contention regarding the critical role of the cen-
tral 7-10 region of NT for this cytoprotective effect was further supported in the present study by our positive findings with [Gln4]NT, and with D-TrpU-NT, a NT analogue with potent hypothermic and neurobehavioral effects u.14. These two NT analogues which bear single changes away from this critical region retained full cytoprotective activity. From the initial experiments using the CRS model, it was clear (vide supra, Table I) that the two N-terminal fragments, NTI_8 and NT1_lO, exhibited significant biological activity when tested at a single dose level. We further examined these fragments by studying dose-response relationships. The cytoprotection afforded by NT1_8 and NTI_I0, like that of NT, was shown to be dose-dependent (Fig. 1) with EDs0, s similar to that of NT (NT = 16.2nmol, NTI_s = 17.8nmol and NTH0 = 19.9 nmol). The parallel nature of the dose-response curves for these 3 peptides suggests that they act at the same receptor site. Binding data using rat brain membrane preparations have shown that the ICs0 values for displacement of [125I]NT binding by NT and certain fragI00
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LOG OF PEPTIDE GONCENTRATION (nmoles) Fig. 1. Dose-response curves for the cytoprotective activity of neurotensin (O), and the N-terminal neurotensin fragments NTI~ (11) and NTl_10 (A). Groups of 24-h food-deprived rats (n = 5-6 rats/dose) were injected i.c. with different concentrations of neurotensin (5.9-17.9 nmol), NTI_8 (5.9-17.9 nmol) and NTl_10 (5.9-17.9 nmol) and the stomachs examined for gastric ulcers after 3 h of CRS. For details see the text. All 3 peptides produced a significant (P < 0.05) dose-dependent cytoprotective response. The correlation coefficients (r) for neurotensin, NTI_8 and NTl_10 are: r = 0.96, r = 0.99 and r = 0.92, respectively.
156 m e n t s of N T (NT2_13, NT4_I3 a n d NT6-13) are very similar, whereas the C - t e r m i n a l f r a g m e n t s (NTs_13 a n d NTg-13) had ICs0, ~ 40-800 fold-higher 19. In m o r e recent work6 it has b e e n d e m o n s t r a t e d that NT6-13 a n d NT4,.13 have an u n u s u a l l y high affinity for rat brain synaptic m e m b r a n e b i n d i n g sites. T h e r e f o r e ,
smaller m o l e c u l a r weight forms of N T that a p p a r e n t ly are n a t u r a l l y occuring m e t a b o l i t e s of the tridecapeptide, i.e. NTI_ s, NTI_10, b u t n o t the C - t e r m i n a l fragments (NTs-13, NT9-13) can effectively exert biological effects. This finding m a y well explain why m a n y effects of centrally a d m i n i s t e r e d N T persist
the p r e s e n t data suggest that these N - t e r m i n a l fragm e n t s m a y utilize a c o m m o n b r a i n N T receptor.
long after the t r i d e c a p e p t i d e has b e e n inactivated.
W h e t h e r the activation of these CNS receptors me-
This research was s u p p o r t e d by N I M H MH-32316, MH-34121, MH-33127 a n d N I C H H D HD-03110.
diates the a f o r e m e n t i o n e d gastric cytoprotective effect is presently u n k n o w n . In conclusion, we have d e m o n s t r a t e d h e r e i n that
1 Aronin, N.,Carraway, R. E., Ferris, C. F., Hammer, R. A. and Leeman, S. E., The stability and metabolism of intravenously administered neurotensin in the rat, Peptides, 3 (1978) 637-642. 2 Bissette, G., Luttinger, D., Mason, G. A., Hernandez, D. E. and Loosen, P. T., Neurotensin and thermoregulation, Ann. N. Y. Acad. Sci., 400 (1982)268-281. 3 Carraway, R. and Leeman, S. E., The isolation of a new bypotensive peptide, neurotensin, from bovine hypothalamus, J. biol. Chem., 248 (1973) 6854--6861. 4 Carraway, R. and Leeman, S. E., Characterization of radioimmunoassayable neurotensin in the rat. Its differential distribution in the central nervous system, small intestine and stomach, J. biol. Chem., 251 (1976) 7045-7052. 5 Dupont, A. and Merand, Y., Enzymatic inactivation of neurotensin by hypothalamic and brain extracts of the rat, Life Sci., 22 (1978) 1263-1630. 6 Granier, G., Van Rietschoten, J., Kitabgi, P., Poustis, G. and Freychet, P., 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, Europ. J. Biochem., 124 (1982) 11%125. 7 Griffiths, E. C., Linton, E. A., McDermott, J. R., Smith, A. I. and Webster, V. A. D., The presence of neurotensininactivating peptidases in rat brain, J. Physiol. (Lond.), 324 (1981) 77 pp. 8 Hernandez, D. E., Nemeroff, C. B. and Prange, A. J., Jr., Ontogeny of the hypothermic response to centrally administered neurotensin in rats, Dev. Brain Res., 3 (1982) 497-501. 9 Hernandez, D. E., Nemeroff, C. B., Orlando, R. C. and Prange, A. J., Jr., Effect of intracisternally administered neuropeptides on the development of stress-induced gastric ulcers in rats, J. Neurosci. Res., (1983) 145-157. 10 Iversen, L. L., Iversen, S. D., Bloom, F. E., Douglas, C., Brown, M. and Vale, W., Calcium-dependent release of somatostatin and neurotensin from rat brain in vitro, Nature (Lond.), 273 (1978) 161-163. 11 Jolicoeur, F. B., Rioux, F., St-Pierre, S. and Barbeau, A., Structure activity of neurotensin's neurobehavioral effects.
W e are grateful to Mrs. Judy M. B a r n e t t a n d Mrs. Dori Y a r b r o u g h for p r e p a r a t i o n of this m a n u s c r i p t .
In R. Collu et al. (Eds.), Brain Peptides, New York, 1982, 171-181. 12 Kitabgi, P., Carraway, R., Van Rietschoten, J., Granier, C., Margot, J. L., Menez, A., Leeman, S. E. and Freychet, P., Neurotensin: specific binding to synaptic membranes from rat brain, Proc. natl. Acad. Sci. U.S.A., 74 (1977) 1846-1850. 13 Lazarus, L. H., Perrin, M. H., Brown, M. R. and Rivier, J. E., Mast cell binding of neurotensin. II. Molecular conformation of neurotensin involved in the stereospecific binding to mast cell receptor sites, J. biol. Chem., 252 (1977) 7180-7183. 14 Loosen, P. T., Nemeroff, C. B., Bissette, G., Burnett, G. B., Prange, A. J., Jr. and Lipton, M. A., Neurotensin-induced hypothermia in the rat: structure activity studies, Neuropharmacology, 17 (1977) 10%113. 15 McDermott, J. R., Smith, A. I., Edwarson, J. A. and Griffiths, E. C., Mechanism of neurotensin degradation by rat brain peptides, Regulat. Peptides, 3 (1982) 397--404. 16 Nemeroff, C. B., Osbahr, A. J. III, Manberg, P. J., Ervin, G. N. and Prange, A. J., Jr., Alterations in nociception and body temperature after intracisternally administered neurotensin, fl-endorphin, other endogenous peptides and morphine, Proc. nat. Acad. Sci. U.S.A., 76 (1979) 5368-5371. t7 Nemeroff, C. B., Hernandez, D. E., Orlando, R. C. and Prange, A. J., Jr., Cytoprotective effect of centrally administered neurotensin on stress-induced gastric ulcers, Amer. J. Physiol., 242 (1982) 342-346. 18 St-Pierre, S., Lalonde, J. M., Gendreau, M., Quirion, R., Regoli, D. and Rioux, F., Synthesis of peptides by the solid phase method. Neurotensin, fragments and analogues, J. med. Chem., 24 (1981) 370-376. 19 Uhl, G. R., Bennett, J. P. and Snyder, S. H., Neurotensin, a central nervous system peptide: apparent receptor binding in brain membranes, Brain Research, 13(J (1977) 29%313. 20 Yamaguchi, T., Tobe, T. and Hikasa, Y., Protection of stress ulcers by infusion of neurotensin, Arch. Jap. Chir.. 59(3) (1980) 269-274.
153
BRE 20213
Evidence for biological activity of two N-terminal fragments of neurotensin, neurotensinl_8 and neurotensinl_10 DANIEL E. HERNANDEZ I, CHARLES M. RICHARDSON l, CHARLES B. NEMEROFF5, ROY C. ORLANDO-', SERGE ST-PIERRE6, FRANCIS RIOUX~and ARTHUR J. PRANGE, Jr. 1.3.4
1Biological Sciences Research Center, Departments ofeMedicine and 3Psychiatry and 4The Neurobiology Program, University of North Carolina School of Medicine, Chapel Hill. NC, 5 Department of Psychiatry, Duke University, Durham, NC ( U. S. A. ) and 6Departments of Physiology and Pharmacology, University of Sherbrooke, Sherbrooke, Que. (Canada) (Accepted January 31st, 1984)
Key words: neurotensin- - neurotensin-fragments - - gastric - - cytoprotection
Intracisternal (i.c.) administration of the endogenous tridecapeptide neurotensin (NT) has been previously shown to significantly reduce the incidence of cold-restraint stress (CRS)-induced gastric ulcers in rats. In this study we confirm the cytoprotective activity of central NT, and document structure-activity relationships for this effect of NT. When tested in a dose equimolar to 17.9 nmol of NT the NT analogs [GIn4]NT, D-TrpII-NT, and D-ArgS-NTwere cytoprotective, whereas o-Argg-NT was not. Gonadotropin-releasing hormone (Gn-RH) and melanocyte-stimulating hormone-release inhibiting factor (MIF-1), two oligopeptides structurally unrelated to NT exhibited no cytoprotective efficacy in this paradigm. The C-terminal fragments of NT, xenopsin and NT8_13, and the N-terminal fragment NTI_6 were completely ineffective. Finally, NT I ~ and NTH0, two N-terminal fragments of NT produced significant cytoprotective activity at this dose level. The cytoprotection afforded by NTI~ and NTH0, like that of NT, was dose-dependent with EDs0's similar to that of NT (NT = 16.2 nmol, NTt_s = 17.8 nmol and NT~ l0 = 19.9 nmol). In conclusion, we demonstrate that smaller molecular weight forms of NT thought to be degradation products of NT can effectively exert biological effects.
Neurotensin (NT), a tridecapeptide ( p G l u - L e u Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-lle-LeuOH), was first isolated from bovine hypothalami 3. Immunohistochemical and radioimmunological evidence has revealed a widespread distribution of NT in the central nervous system (CNS) and in n o n - n e u ral peripheral tissues, including the gastrointestinal tract, of several vertebrate species 4. In the CNS, N T has been found in the synaptosomal fraction after density gradient centrifugation, and specific high-affinity receptors for NT have been identified in brain m e m b r a n e s 12. Further, depolarizing stimuli have been shown to produce a calciumdependent release of immunoreactive NT from brain tissue slices, suggesting that N T may serve a role in neurotransmission ~0. Consistent with this hypothesis is the considerable body of evidence indicating that
brain NT may play a physiological role in thermoregulation 2.s, antinociception 16, and m a i n t e n a n c e of gastric mucosal integrity iT. These effects of NT appear to be mediated within the CNS, because peripheral administration of NT is devoid of either hypothermic, antinociceptive or cytoprotective activity9A7. However, NT-induced cytoprotection has also been observed after peripheral injection in the restraintwater immersion test 20. Degradation of N T by peptidases has been demonstrated in brain as well as in peripheral plasmal,5, 7A5. After in vitro incubation in a rat brain m e m b r a n e preparation, the main product in both soluble and particulate fractions are NTI_8 and the C-terminal fragment NTg_13. In addition, the Nterminal fragment NTI_8 appears to be relatively resistant to further degradation, whereas NT9_13, in contrast, is rapidly degradated to liberate tyrosine as
Correspondence: D. E. Hernandez, Biological Sciences Research Center 220-H, University of North Carolina School of Medicine, Chapel Hill, NC 27514, U.S.A. 0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V.
154 a result of a post-proline cleavage at Pro l0 (ref. 15). The decapeptide N-terminal fragment (NTH0) has also been identified after in vivo incubation, but proportionally more of this decapeptide can be identified from the particulate, rather than the soluble fraction'S. The results of incubation of NT in rat plasma have also been studied in some detail. Intravenous injection of radiolabeled [3H]NT has been shown to generate NTI_8, NTl_ll and NT9_13. The apparent halflives of intravenously injected synthetic NTI_s and NT1_I1 are 9.5 and 5 min, respectively, while that of NTg-13 is less than 0.5 min~. In this report we demonstrate that two N-terminal fragments of NT, NTI_s and NTl_10, exert significant cytoprotective activity as measured by their ability to significantly prevent the development of cold-restraint-stress (CRS)-induced gastric ulcers in rats after intracisternal (i.c.) injection. Adult male Sprague-Dawley rats (220-250 g) from Zivic-Miller (Cincinnati, OH) were kept in an environmentally controlled room on a 12 h dark: 12 h light schedule (lights on 06.00 h), and fed laboratory chow and water ad libitum. They were maintained in the animal facility for at least one week prior to experimental use. All rats were deprived of food but not water 24 h prior to the experiments. Peptides or vehicle (10 ¢tl of 0.9% NaCI) were administered i.c. under light ether anesthesia as described elsewhere 17. Immediately after the i.c. injection animals were restrained in wire mesh and placed supine in a cold (4 °C) room for 3 h. This regimen of physical restraint and simultaneous exposure to a cold environment has been reported previously to reliably produce ulcers in the glandular portion of the stomach of food-deprived rats 9.17. Following the 3 h stress period, rats were killed by decapitation and the stomach examined for gastric ulcerations by a trained observer with no knowledge of the treatment regimen. The presence of gastric ulcers was assessed by using the following scale: 0, no pathology; 1+ mucosal edema and/or petechiae; and 2+ gross gastric mucosal ulceration. The results, which are expressed as cytoprotective response, represent the percentage of rats that exhibited no gastric lesions. In the first series of experiments all substances were initially administered i.c. in a dose equimolar to 17.9 nmol of NT. This dose of NT was chosen be-
cause it has been previously demonstrated to produce a significant cytoprotective response in this model 9.17. The cytoprotective response of each treatment group was compared to that of the saline-treated controls. Statistical significance was assessed with the Kruskal-Wailis test for multiple comparisons of non-parametric data. Because the two NT fragments, NT~_8 and NTI_I0, elicited an almost identical cytoprotective effect when tested at a single dose level as did NT itself, dose-response studies were performed by administering different doses of NT and these two NT fragments to allow direct comparison with the parent compound (NT). The dose-response curves were generated with the least-square method, followed by simple linear regression analysis; P values for the curves were determined with the Chi-square test. In addition, the EDso for NT and both NT fragments was determined from the dose-response curves using the probit procedure. In all the statistical tests, P values of 0.05 or less were considered to represent significant differences between treatment groups. Neurotensin, xenopsin, gonadotropin-releasing hormone (Gn-RH) and melanocyte-stimulating hormone-release-inhibiting-factor (MIF-I) were obtained from Bachem (Torrance, CA). Two NT fragments, NT~_6 and NTI_~, were purchased from Cambridge Research Biochemicals Ltd. (Harston, Cambridgeshire, U.K.), and [Gin4]neurotensin from Peninsula Laboratories (San Carlos, CA). The following structural analogues of NT: DArgS-NT, D-Arg9-NT, D-Trp I t-NT and the NT fragments, NTs_E3and NT 1 l/~, were synthetized by one of us (S.St-P), using solid phase methodology as previously described TM. The results obtained in the first set of experiments, which are presented in Table I, show that, as previously demonstratedg,l 7. i.c. NT (17.9 nmol) produced significant gastric cytoprotection. When tested in a dose equimolar to 17.9 nmol of NT, the N-terminal fragment NTI_6 as well as the C-terminal fragment NTs-13 were not cytoprotective. Furthermore, xenopsin, an octapeptide which closely resembles the C-terminal fragment of NT, and therefore can be considered a natural analogue of NT8-13, was also ineffective. The peptides structurally unrelated to NT including GnRH and M1F-I exhibited no cytoprotective efficacy in this test.
155 TABLE I
Effects of centrally administered peptides on cold-restraint stress (CRS)-induced gastric ulcers in rats All substances were administered intracisternally in a dose equimolar to 17.9 nmol of neurotensin.
Peptide
n
Cytoprotective response
(%) Saline Neurotensin [GIn4]NT o-Trpll-NT D-ArgS-NT NTI_10 NTI~s D-Argg-NT Xenopsin NT8_13 NTv, MIF-I Gn-RH
60 40 10 10 10 13 12 10 9 10 10 10 10
32.3 85* 100"* 100"* 90* 86.7* 75* 60 55.6 50 50. 50 40
** P < 0.001, * P < 0.01 Kruskal-Wallis test (chi-square approximation). All comparisons were done against saline-treated controls.
The fact that NTv.6 and the C-terminal fragments of NT (NT8_13, xenopsin) are ineffective, suggsts the hypothesis that the middle portion of the NT molecule (-Pro7-ArgS-Arg9-Pro 10) appears to play a critical role for the expression of biological activity in this test. Both NT~_8 and NTI_~0 exhibited significant and substantial cytoprotective efficacy at this dose level (Table I). We next examined this hypothesis by testing several NT analogues with single stereoisomeric D-amino acid replacements for the naturally occurring Lforms. The fact that D-ArgS-NT, but not D-Arg9-NT produced a significant cytoprotective response similar to NT, indicates that within this critical region there appears to be some degree of conformational specificity. These results are concordant with findings pertaining to hypothermi# 4. From structure-activity studies it has been reported that D-Arg9-NT, and the C-terminal fragment NT9._13, but not DArgS-NT, are devoid of hypothermic activity. Furthermore, systematic substitution of the arginine residues at position 8 and 9 in NT by L-Ala have revealed that both arginine residues are required for optimal recognition by mast cell receptors, and it has been proposed that Arg 8 plays a greater role than Arg 9 in this particular radioreceptor assay 13. This contention regarding the critical role of the cen-
tral 7-10 region of NT for this cytoprotective effect was further supported in the present study by our positive findings with [Gln4]NT, and with D-TrpU-NT, a NT analogue with potent hypothermic and neurobehavioral effects u.14. These two NT analogues which bear single changes away from this critical region retained full cytoprotective activity. From the initial experiments using the CRS model, it was clear (vide supra, Table I) that the two N-terminal fragments, NTI_8 and NT1_lO, exhibited significant biological activity when tested at a single dose level. We further examined these fragments by studying dose-response relationships. The cytoprotection afforded by NT1_8 and NTI_I0, like that of NT, was shown to be dose-dependent (Fig. 1) with EDs0, s similar to that of NT (NT = 16.2nmol, NTI_s = 17.8nmol and NTH0 = 19.9 nmol). The parallel nature of the dose-response curves for these 3 peptides suggests that they act at the same receptor site. Binding data using rat brain membrane preparations have shown that the ICs0 values for displacement of [125I]NT binding by NT and certain fragI00
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I 1.0
I 1.5
LOG OF PEPTIDE GONCENTRATION (nmoles) Fig. 1. Dose-response curves for the cytoprotective activity of neurotensin (O), and the N-terminal neurotensin fragments NTI~ (11) and NTl_10 (A). Groups of 24-h food-deprived rats (n = 5-6 rats/dose) were injected i.c. with different concentrations of neurotensin (5.9-17.9 nmol), NTI_8 (5.9-17.9 nmol) and NTl_10 (5.9-17.9 nmol) and the stomachs examined for gastric ulcers after 3 h of CRS. For details see the text. All 3 peptides produced a significant (P < 0.05) dose-dependent cytoprotective response. The correlation coefficients (r) for neurotensin, NTI_8 and NTl_10 are: r = 0.96, r = 0.99 and r = 0.92, respectively.
156 m e n t s of N T (NT2_13, NT4_I3 a n d NT6-13) are very similar, whereas the C - t e r m i n a l f r a g m e n t s (NTs_13 a n d NTg-13) had ICs0, ~ 40-800 fold-higher 19. In m o r e recent work6 it has b e e n d e m o n s t r a t e d that NT6-13 a n d NT4,.13 have an u n u s u a l l y high affinity for rat brain synaptic m e m b r a n e b i n d i n g sites. T h e r e f o r e ,
smaller m o l e c u l a r weight forms of N T that a p p a r e n t ly are n a t u r a l l y occuring m e t a b o l i t e s of the tridecapeptide, i.e. NTI_ s, NTI_10, b u t n o t the C - t e r m i n a l fragments (NTs-13, NT9-13) can effectively exert biological effects. This finding m a y well explain why m a n y effects of centrally a d m i n i s t e r e d N T persist
the p r e s e n t data suggest that these N - t e r m i n a l fragm e n t s m a y utilize a c o m m o n b r a i n N T receptor.
long after the t r i d e c a p e p t i d e has b e e n inactivated.
W h e t h e r the activation of these CNS receptors me-
This research was s u p p o r t e d by N I M H MH-32316, MH-34121, MH-33127 a n d N I C H H D HD-03110.
diates the a f o r e m e n t i o n e d gastric cytoprotective effect is presently u n k n o w n . In conclusion, we have d e m o n s t r a t e d h e r e i n that
1 Aronin, N.,Carraway, R. E., Ferris, C. F., Hammer, R. A. and Leeman, S. E., The stability and metabolism of intravenously administered neurotensin in the rat, Peptides, 3 (1978) 637-642. 2 Bissette, G., Luttinger, D., Mason, G. A., Hernandez, D. E. and Loosen, P. T., Neurotensin and thermoregulation, Ann. N. Y. Acad. Sci., 400 (1982)268-281. 3 Carraway, R. and Leeman, S. E., The isolation of a new bypotensive peptide, neurotensin, from bovine hypothalamus, J. biol. Chem., 248 (1973) 6854--6861. 4 Carraway, R. and Leeman, S. E., Characterization of radioimmunoassayable neurotensin in the rat. Its differential distribution in the central nervous system, small intestine and stomach, J. biol. Chem., 251 (1976) 7045-7052. 5 Dupont, A. and Merand, Y., Enzymatic inactivation of neurotensin by hypothalamic and brain extracts of the rat, Life Sci., 22 (1978) 1263-1630. 6 Granier, G., Van Rietschoten, J., Kitabgi, P., Poustis, G. and Freychet, P., 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, Europ. J. Biochem., 124 (1982) 11%125. 7 Griffiths, E. C., Linton, E. A., McDermott, J. R., Smith, A. I. and Webster, V. A. D., The presence of neurotensininactivating peptidases in rat brain, J. Physiol. (Lond.), 324 (1981) 77 pp. 8 Hernandez, D. E., Nemeroff, C. B. and Prange, A. J., Jr., Ontogeny of the hypothermic response to centrally administered neurotensin in rats, Dev. Brain Res., 3 (1982) 497-501. 9 Hernandez, D. E., Nemeroff, C. B., Orlando, R. C. and Prange, A. J., Jr., Effect of intracisternally administered neuropeptides on the development of stress-induced gastric ulcers in rats, J. Neurosci. Res., (1983) 145-157. 10 Iversen, L. L., Iversen, S. D., Bloom, F. E., Douglas, C., Brown, M. and Vale, W., Calcium-dependent release of somatostatin and neurotensin from rat brain in vitro, Nature (Lond.), 273 (1978) 161-163. 11 Jolicoeur, F. B., Rioux, F., St-Pierre, S. and Barbeau, A., Structure activity of neurotensin's neurobehavioral effects.
W e are grateful to Mrs. Judy M. B a r n e t t a n d Mrs. Dori Y a r b r o u g h for p r e p a r a t i o n of this m a n u s c r i p t .
In R. Collu et al. (Eds.), Brain Peptides, New York, 1982, 171-181. 12 Kitabgi, P., Carraway, R., Van Rietschoten, J., Granier, C., Margot, J. L., Menez, A., Leeman, S. E. and Freychet, P., Neurotensin: specific binding to synaptic membranes from rat brain, Proc. natl. Acad. Sci. U.S.A., 74 (1977) 1846-1850. 13 Lazarus, L. H., Perrin, M. H., Brown, M. R. and Rivier, J. E., Mast cell binding of neurotensin. II. Molecular conformation of neurotensin involved in the stereospecific binding to mast cell receptor sites, J. biol. Chem., 252 (1977) 7180-7183. 14 Loosen, P. T., Nemeroff, C. B., Bissette, G., Burnett, G. B., Prange, A. J., Jr. and Lipton, M. A., Neurotensin-induced hypothermia in the rat: structure activity studies, Neuropharmacology, 17 (1977) 10%113. 15 McDermott, J. R., Smith, A. I., Edwarson, J. A. and Griffiths, E. C., Mechanism of neurotensin degradation by rat brain peptides, Regulat. Peptides, 3 (1982) 397--404. 16 Nemeroff, C. B., Osbahr, A. J. III, Manberg, P. J., Ervin, G. N. and Prange, A. J., Jr., Alterations in nociception and body temperature after intracisternally administered neurotensin, fl-endorphin, other endogenous peptides and morphine, Proc. nat. Acad. Sci. U.S.A., 76 (1979) 5368-5371. t7 Nemeroff, C. B., Hernandez, D. E., Orlando, R. C. and Prange, A. J., Jr., Cytoprotective effect of centrally administered neurotensin on stress-induced gastric ulcers, Amer. J. Physiol., 242 (1982) 342-346. 18 St-Pierre, S., Lalonde, J. M., Gendreau, M., Quirion, R., Regoli, D. and Rioux, F., Synthesis of peptides by the solid phase method. Neurotensin, fragments and analogues, J. med. Chem., 24 (1981) 370-376. 19 Uhl, G. R., Bennett, J. P. and Snyder, S. H., Neurotensin, a central nervous system peptide: apparent receptor binding in brain membranes, Brain Research, 13(J (1977) 29%313. 20 Yamaguchi, T., Tobe, T. and Hikasa, Y., Protection of stress ulcers by infusion of neurotensin, Arch. Jap. Chir.. 59(3) (1980) 269-274.