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Biological activity of neurotensin and its C-terminal partial sequences
Brain Research
Bulletin,
Vol. 3, pp. 555-557. Printed in the U.S.A.
BRIEF COMMUNICATION Biological Activity of Neurotensin and its C-Terminal Partial Sequences KIYOSHI
KATAOKA,
Department
ATARU TANIGUCHI, HIDEKAZU SHIMIZU, KENJI SODA, SACHIKO HARUAKI YAJIMA’ AND KOKI KITAGAWA*
of Physiology,
Ehime University School of Medicine, (Received
Onsen-gun,
OKUNO
Ehime 791-02, Japan
24 January 1978)
KATAOKA, K., A. TANIGUCHI,
H. SHIMIZU, K. SODA, S. OKUNO, H. YAJIMA AND K. KITAGAWA. Biological partial sequences. BRAIN RES. BULL. 3(5) 555-557, 1978.-A hypothalamic tridecapeptide, neurotensin, and its C-terminal partial sequences down to the dipeptide were synthesized. These peptides were assayed for smooth muscle contracting and blood pressure lowering properties using preparations of isolated stomach fundus, uterus and duodenum of the rat, isolated guinea pig ileum and rabbit carotid artery. Sequences of 6 or more terminal amino acids produced a strong stomach fundus contracting effect, the potencies of the fragments being approximately equivalent to or slightly more than that of the parent tridecapeptide. These fragments also elicited the ileum contracting activity, but the potencies were only one fifth to one tenth that of neurotensin. The tetra- or dipeptide hardly stimulated either the fundus or the ileum. The stimulating effect on the uterus or relaxing effect on the duodenum of neurotensin was not consistent. Rabbit blood pressure was lowered markedly by neurotensin and weakly by its dodecapepide. From these results, the arginine or arginine-arginine residue down to the C-terminal leucine appears to be essential for the smooth muscle contracting activity of neurotensin. The full length sequence may be needed for the hypotensive effect. Neurotensin C-terminal sequence Biological activity Rat stomach fundus Guinea pig ileum Rabbit blood pressure
activity
of neurotensin
and its C-terminal
NEUROTENSIN, a tridecapeptide (pyrGlu-Leu-Tyr-GlnAsn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-OH) isolated from bovine hypothalamus [3,4], has potent pharmacological effects both in vivo and in vitro. Peripherally administered neurotensin elicits hypotension, increased vascular permeability and hyperglycemia [4,5]. In vitro, this peptide induces contraction of isolated guinea pig ileum and relaxation of isolated rat duodenum [5]. In the central nervous system, regionally specific distribution [8] and subcellularly synaptosomal localization [ 121 of, and occurrence of receptors [ 131 specific for neurotensin were already elucidated. Further, direct central actions of neurotensin have been demonstrated; namely, it lowers body temperature [2] and accentuates barbiturate sedation [lo]. These results strongly suggest that neurotensin may be a specific neuropeptide and is likely to function as a transmitter. Thus, it appears to be important to clarify the biologically active structure of the neurotensin molecule, and some findings thereof have already been reported [6,11]. The present report describes an extensive correlative study of the biological activities of neurotensin with its chain length, using the C-terminal partial sequences down to the dipeptide. A finding of a sensitive contractility of rat stomach fundus to the trideca-, down to the hexapeptide is principally mentioned.
METHOD
Neurotensin was synthesized as described previously [15]. Briefly, 5 dipeptide units, Ile-Leu, Pro-Tyr, Arg-Arg, Lys-Pro and Leu-Tyr were condensed successively. Asn, Gln and pyrGlu residues were also introduced stepwise. In this way, C-terminal di-, tetra-, hexa-, octa-, nona-, decaand dodecapeptide fragments were obtained during synthesis of the whole neurotensin molecule. Rat stomach fundus, about 3 cm long segments of the longitudinal muscle [14], were suspended in 5 ml organ bath containing aerated Krebs-Ringer’s solution at 30°C. Segments of distal guinea pig ileum, 1.5-2 cm in length, were suspended in a 5 ml organ bath containing aerated Tyrode’s solution at 30°C. Rat uterus segments at oesterous stage, 1.5 cm in length, and rat duodenum segments, 1.5-2 cm length, were treated in the same manner as that for the fundus. Contractions of these smooth muscle preparations were monitored isotonically by a smooth muscle transducer (Nihon-Koden, SB-IT) coupled to an inkwriter, using a load of 0.25-1.0 g, and recorded. The blood pressure of rabbits of under both sexes, weighing 2-2.5 Kg, was recorded, urethane anaesthesia, through a catheter inserted in a carotid artery connected to a blood pressure transducer. Peptides to be analysed were dissolved in saline, and the volume
‘Faculty of Pharmaceutical Sciences, Kyoto University, ?Faculty of Pharmaceutical
Kyoto 606, Japan. Sciences, Tokushima University, Tokushima 770, Japan.
Copyright 0 1978 ANKHO
International Inc .-036
l -9230/78/050555-03$00.80/O
556
KATAOKA TABLE
ET AL.
1
RELATIVE GUT CONTRACTING POTENCIES OF THE PARTIAL SEQUENCES TO THAT OF NEUROTENSIN Amino Acid ~eurotensin Dodecapeptide Decapeptide Nonapeptide Octapeptide Hexapeptide Tetrapeptide Dipeptide
Sequence
pyffilu-LRu-Tyr-Gln-Asn-Lys-Pro_Arg-Arg-Pro-Tyr-Ile-Leu~OH) Leu-Tyr-Gin-Asn-Lys-~~Arg-Arg-ho-Tar-Ile-LeutOH) Gin-Asn-Lys-do-Arg~Arg~~o-Tyr-Ile-Leu(G~) Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Be-Leu(OH) l~ys~Pr~Ar~Arg~~o-Tyr-Ile-Leu(OH) Arg-Arg-Pro-Tyr-Ile-Leu(OII) Pro-Tyr-Ile-Leu(OH)
be-LeuiOH)
Rat Stomach Fundus
Guinea Pig Ileum
100 m-110
20-30
90-140 90-140 90-140 7tl-loo is C5
105 20-30 lo-30 l@-20 l&20
Full dose-response curves were recorded for all the peptides. The slopes were effectively parallel for the sequences down to the hexaneotide. The netencies relative to neurotensin (accented as iOO) were expressed as the ratios of doses fnr each peptide which _ produced half maximal cont~ction.
applied was limited to 0.1 ml either for organ bath or IV injection through the femoral vein. RESUCI‘S AND DISCUSSION
Neurotensin had the greatest effect on rat stomach fundus, eliciting contraction at. the threshold concent~tion of 2x10 111M and that of the half maximal response at 3 x I@ * M, respectively. The sequences down to the hexapeptide also showed appreciable activity and it should be noted here that the o&a-, nona- and decapeptide had up to 1.4 times higher activity than neurotensin (Table 1). The tetra- and dipeptide were negli~ibIy active. From these results, it can be stated that the Arg residue or Arg-Arg residue at the C-terminal sequences is essential for the activity. The finding that the activity was not provisional to their chain length indicates a possibility that a couple of amino acids at the N-terminal may modify the structure and hence the stability of the neurotensin molecule. There is a dissociation between the relative pharmacological and relative receptor binding potencies of the sequences. In the binding assay, the octapeptide is about one half, the hexapeptide one tenth and the pentapeptid~ only one two hundredth active as neurotensin 113f. This suggests the requirement of at least nine amino acids of the C-terminal sequence for receptor binding of neurotensin, a sharp contrast with that for the fundus st~mulatjon. These contractions were hardly affected by the addition to the bathing medium of atropine (lo-” g/ml), tryptamine (IO i g/ml) and piribenzamine [RI-” g/ml), a finding which indicated that neurotensin stimulated directly the muscle through specific binding to neurotensin receptors, but unlikely to interact with cholinergic, serotonergic or histaminer~i~ mech~ism in the stomach fundus. This smooth muscle preparation was found to be easily desensitized by successive large scale application of the peptides, so that amounts of samples to be tested were to be limited to not more than those eliciting submaximal contraction and addition to the bath was to be made at least 4 min after its prior administration and repeated washings. It has already been demonstrated that the rat stomach fundus is highiy sensitive to 5-hydroxytryptamine 1141 and hence has long been employed as a test object for bioassay of this amine. According to our earlier investigation 171, this preparation was also induced contraction by pa~ially purified substance P. and this
was confirmed again in the present experiment in which synthetic substance P [9] at the concentration of 4-5 x lo-!’ M elicited half maximal contraction of the muscle, a finding indicating a little less efIicacy of substance P to the fundus than neurotensin. It is interesting to note here that xenopsin, an o~tapeptide isolated from frog skin having ~e~rotensin like structure, pyrGlu-Gly-Lys”Arg-pro-Tyr-Ile-Leu~OH), atso showed a rat stomach fundus stimulating activity [I I. Earlier authors reported 1.5,6, 1I] that an isolated guinea pig ileum was also sensitive to neurotensin and contracts. This was confirmed in the present study. The minima1 effective concentration and the mean concentration required to cause the half maximal contraction was 5x 1W”’ M and 1.5x IO-x M, respectively. C-terminal sequences down to the hexapeptide all showed similar activities of one fifth to one tenth that of neurotensin. The tetra- and dipeptide did not have any appreciable activity at concentrations of up to 5~ IO-” M, indicating again that the Arg or Arg-Arg residue is essential for the smooth muscle contraction. The pyrGlu residue at the N-terminal is also essential for the full activity of neurotensin on this test object, a contrast with the stomach fundus. Segments of rat uterus, regardless of their hormonal stage, responded to neurotensin, but their sensitivities were variable and much less than those of rat stomach fundus and the guinea pig ileum, so that they were not studied in detail. In contrast and confirming the earlier reports [ 51, rat duodenum showed relaxation to neurotensin , but the extent of this relaxation was slight and variable, and hence dose-response relation was unclear. It can be said, therefore, that the uterus or duodenum is not a suitable tissue for neurote~sin bioassay. All these in vitro preparations showed prompt and intense ta~hyphylax~s and unaffectiveness by atrapine. tryptamine and pitibenzamine, similar findings to the rat stomach fundus. Intravenous injection of IO0 nmoles of neurotensin induced a considerable fall in the blood pressure of the rabbit for OS-2 min with practically no aiteration in the heart rate. The effect of the dodecapeptide or shorter peptides were markedly weak on the blood pressure, suggesting that the full length of the molecule is needed to elicit the activity. Desensitization was also marked: however, successive application was possible after a considerable intervals of more than 20 min. Recent rapid progress in neuropeptide research is Iargely due to the development of methods using antiserum to a hormone in question, and in most research hormones are
BIOLOGICAL
ACTIVITY
OF NEUROTENSIN
FRAGMENTS
analysed by simple radioimmunoassay or receptor binding assay. However, localization of active center for antibody binding, receptor binding or pharmacological activity is not always identical and consequently it can be possible that values obtained by different procedures differ significantly. This leads to the necessity of parallel different assays includ-
ing tedious bioassay for certain cases of peptide hormone research. In this connexion, the rat stomach fundus may be a good tool for in vitro bioassay of neurotensin, although elimination of interaction by the fundus active contaminants in brain tissue extract such as S-hydroxytryptamine or substance P should be carefully made in the assay system.
REFERENCES Tachibana, M. Uchiyama, T. Nakajima and T. Yasuhara. Isolation and structure of a new active peptide “Xenopsin” on the smooth muscle, especially on a strip of fun-
8. Kobayashi, R. M., M. Brown and W. Vale. Regional distribution of neurotensin and somatostatin in rat brain. Bruin Res.
dus from a rat stomach, from the skin ofXenopus laevis. Chem. Pharmac. Bull. 21: 2801-2804, 1973. Bissette, Cl., C. B. Nemeroff, P. T. Loosen, A. J. Prange, Jr. and M. A. Lipton. Hypothermia and intolerance to cold induced by intracistemal administration of the hypothalamic peptide neurotensin. Nature (Land.) 262: 607-609, 1976. Carraway, R. and S. E. Leeman. The isolation of a new hypothalamic peptide, neurotensin, from bovine hypothalami. J. hiol. Chem. 248: 6854-6861, 1973. Carraway, R. and S. E. Leeman. The amino acid sequence of a hypothalamic peptide, neurotensin. J. hiol. Chem. 250: 19071911, 1975. Carraway, R. and S. E. Leeman. The synthesis of neurotensin. J. biol. Chem. 250: 1912-1918, 1975. Carraway, R. and S. E. Leeman. Structural requirements for the biological activity of neurotensin, a new vasoactive peptide. In: Peptides: Chemistry, Structure und Biology, edited by R. Walter and J. Meinhofer. Ann Arbor: Ann Arbor Science Publishers, 1975, pp. 679-685. Kataoka, K. Subcellular distribution of 5-hydroxytryptamine in the rabbit brain. Jap. J. Physiol. 12: 623-638, 1%2.
Substance P. Life SC;. 15: 2033-2044, 1974. 10. Prange, A. J., Jr., G. R. Breese, G. D. Jahnke, B. R. Martin, B. R. Cooper, J. M. Cott, I. C. Wilson, L. B. Alltop, M. A. Lipton, G. Bissette, C. B. Nemeroff and P. T. Loosen. Modification of pentobarbital effects by natural and synthetic polypeptides: dissociation of brain and pituitary effect. Life Sci. 16: 1907-1914,
1. Araki, K., S.
2.
3.
4.
5. 6.
7.
126: 58&588, 1977. 9. Leeman, S. E. and E. A. Mroz. Minireview.
1975.
11. Segawa, T., M. Hosokawa, K. Kitagawa and H. Yajima. Contractile activity of synthetic neurotensin and related polypeptides on guinea pig ileum. J. Pharm. Pharmuc. 29: 57-58, 1977. 12 Uhl, G. R. and S. H. Snyder. Regional and subcellular distribution of brain neurotensin. Life Sci. 19: 1827-1832, 1976. 13 Uhl, G. R., J. P. Bennett, Jr. and S. H. Snyder. Neurotensin, a central nervous system peptide: apparent receptor binding in brain membranes. Brcrin Res. 130: 299-313, 1977. 14. Vane, J. R. A sensitive method for the assay of 5-hydroxytryptamine. Br. J. Phrrrmtrc. 12: 344-349, 1957. 15. Yajima, H., K. Kitagawa, T. Segawa, M. Nakano and K. Kataoka. Studies on peptides. Application of trifluoromethanesulphonic acid as a deblocking reagent to the synthesis of neurotensin. Chem. pharmcrc~. Bull. 23: 3299-3301, 1975.
Note Added in Proof
After submitting the manuscript, a report by A. Rokaeus et al., (Actu phurmac. tax. 41: 141-147, 1!377) appeared, demonstrating findings similar to the present results; that neurotensin has a potent stimulating activity on isolated rat stomach fundus.
Bulletin,
Vol. 3, pp. 555-557. Printed in the U.S.A.
BRIEF COMMUNICATION Biological Activity of Neurotensin and its C-Terminal Partial Sequences KIYOSHI
KATAOKA,
Department
ATARU TANIGUCHI, HIDEKAZU SHIMIZU, KENJI SODA, SACHIKO HARUAKI YAJIMA’ AND KOKI KITAGAWA*
of Physiology,
Ehime University School of Medicine, (Received
Onsen-gun,
OKUNO
Ehime 791-02, Japan
24 January 1978)
KATAOKA, K., A. TANIGUCHI,
H. SHIMIZU, K. SODA, S. OKUNO, H. YAJIMA AND K. KITAGAWA. Biological partial sequences. BRAIN RES. BULL. 3(5) 555-557, 1978.-A hypothalamic tridecapeptide, neurotensin, and its C-terminal partial sequences down to the dipeptide were synthesized. These peptides were assayed for smooth muscle contracting and blood pressure lowering properties using preparations of isolated stomach fundus, uterus and duodenum of the rat, isolated guinea pig ileum and rabbit carotid artery. Sequences of 6 or more terminal amino acids produced a strong stomach fundus contracting effect, the potencies of the fragments being approximately equivalent to or slightly more than that of the parent tridecapeptide. These fragments also elicited the ileum contracting activity, but the potencies were only one fifth to one tenth that of neurotensin. The tetra- or dipeptide hardly stimulated either the fundus or the ileum. The stimulating effect on the uterus or relaxing effect on the duodenum of neurotensin was not consistent. Rabbit blood pressure was lowered markedly by neurotensin and weakly by its dodecapepide. From these results, the arginine or arginine-arginine residue down to the C-terminal leucine appears to be essential for the smooth muscle contracting activity of neurotensin. The full length sequence may be needed for the hypotensive effect. Neurotensin C-terminal sequence Biological activity Rat stomach fundus Guinea pig ileum Rabbit blood pressure
activity
of neurotensin
and its C-terminal
NEUROTENSIN, a tridecapeptide (pyrGlu-Leu-Tyr-GlnAsn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu-OH) isolated from bovine hypothalamus [3,4], has potent pharmacological effects both in vivo and in vitro. Peripherally administered neurotensin elicits hypotension, increased vascular permeability and hyperglycemia [4,5]. In vitro, this peptide induces contraction of isolated guinea pig ileum and relaxation of isolated rat duodenum [5]. In the central nervous system, regionally specific distribution [8] and subcellularly synaptosomal localization [ 121 of, and occurrence of receptors [ 131 specific for neurotensin were already elucidated. Further, direct central actions of neurotensin have been demonstrated; namely, it lowers body temperature [2] and accentuates barbiturate sedation [lo]. These results strongly suggest that neurotensin may be a specific neuropeptide and is likely to function as a transmitter. Thus, it appears to be important to clarify the biologically active structure of the neurotensin molecule, and some findings thereof have already been reported [6,11]. The present report describes an extensive correlative study of the biological activities of neurotensin with its chain length, using the C-terminal partial sequences down to the dipeptide. A finding of a sensitive contractility of rat stomach fundus to the trideca-, down to the hexapeptide is principally mentioned.
METHOD
Neurotensin was synthesized as described previously [15]. Briefly, 5 dipeptide units, Ile-Leu, Pro-Tyr, Arg-Arg, Lys-Pro and Leu-Tyr were condensed successively. Asn, Gln and pyrGlu residues were also introduced stepwise. In this way, C-terminal di-, tetra-, hexa-, octa-, nona-, decaand dodecapeptide fragments were obtained during synthesis of the whole neurotensin molecule. Rat stomach fundus, about 3 cm long segments of the longitudinal muscle [14], were suspended in 5 ml organ bath containing aerated Krebs-Ringer’s solution at 30°C. Segments of distal guinea pig ileum, 1.5-2 cm in length, were suspended in a 5 ml organ bath containing aerated Tyrode’s solution at 30°C. Rat uterus segments at oesterous stage, 1.5 cm in length, and rat duodenum segments, 1.5-2 cm length, were treated in the same manner as that for the fundus. Contractions of these smooth muscle preparations were monitored isotonically by a smooth muscle transducer (Nihon-Koden, SB-IT) coupled to an inkwriter, using a load of 0.25-1.0 g, and recorded. The blood pressure of rabbits of under both sexes, weighing 2-2.5 Kg, was recorded, urethane anaesthesia, through a catheter inserted in a carotid artery connected to a blood pressure transducer. Peptides to be analysed were dissolved in saline, and the volume
‘Faculty of Pharmaceutical Sciences, Kyoto University, ?Faculty of Pharmaceutical
Kyoto 606, Japan. Sciences, Tokushima University, Tokushima 770, Japan.
Copyright 0 1978 ANKHO
International Inc .-036
l -9230/78/050555-03$00.80/O
556
KATAOKA TABLE
ET AL.
1
RELATIVE GUT CONTRACTING POTENCIES OF THE PARTIAL SEQUENCES TO THAT OF NEUROTENSIN Amino Acid ~eurotensin Dodecapeptide Decapeptide Nonapeptide Octapeptide Hexapeptide Tetrapeptide Dipeptide
Sequence
pyffilu-LRu-Tyr-Gln-Asn-Lys-Pro_Arg-Arg-Pro-Tyr-Ile-Leu~OH) Leu-Tyr-Gin-Asn-Lys-~~Arg-Arg-ho-Tar-Ile-LeutOH) Gin-Asn-Lys-do-Arg~Arg~~o-Tyr-Ile-Leu(G~) Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Be-Leu(OH) l~ys~Pr~Ar~Arg~~o-Tyr-Ile-Leu(OH) Arg-Arg-Pro-Tyr-Ile-Leu(OII) Pro-Tyr-Ile-Leu(OH)
be-LeuiOH)
Rat Stomach Fundus
Guinea Pig Ileum
100 m-110
20-30
90-140 90-140 90-140 7tl-loo is C5
105 20-30 lo-30 l@-20 l&20
Full dose-response curves were recorded for all the peptides. The slopes were effectively parallel for the sequences down to the hexaneotide. The netencies relative to neurotensin (accented as iOO) were expressed as the ratios of doses fnr each peptide which _ produced half maximal cont~ction.
applied was limited to 0.1 ml either for organ bath or IV injection through the femoral vein. RESUCI‘S AND DISCUSSION
Neurotensin had the greatest effect on rat stomach fundus, eliciting contraction at. the threshold concent~tion of 2x10 111M and that of the half maximal response at 3 x I@ * M, respectively. The sequences down to the hexapeptide also showed appreciable activity and it should be noted here that the o&a-, nona- and decapeptide had up to 1.4 times higher activity than neurotensin (Table 1). The tetra- and dipeptide were negli~ibIy active. From these results, it can be stated that the Arg residue or Arg-Arg residue at the C-terminal sequences is essential for the activity. The finding that the activity was not provisional to their chain length indicates a possibility that a couple of amino acids at the N-terminal may modify the structure and hence the stability of the neurotensin molecule. There is a dissociation between the relative pharmacological and relative receptor binding potencies of the sequences. In the binding assay, the octapeptide is about one half, the hexapeptide one tenth and the pentapeptid~ only one two hundredth active as neurotensin 113f. This suggests the requirement of at least nine amino acids of the C-terminal sequence for receptor binding of neurotensin, a sharp contrast with that for the fundus st~mulatjon. These contractions were hardly affected by the addition to the bathing medium of atropine (lo-” g/ml), tryptamine (IO i g/ml) and piribenzamine [RI-” g/ml), a finding which indicated that neurotensin stimulated directly the muscle through specific binding to neurotensin receptors, but unlikely to interact with cholinergic, serotonergic or histaminer~i~ mech~ism in the stomach fundus. This smooth muscle preparation was found to be easily desensitized by successive large scale application of the peptides, so that amounts of samples to be tested were to be limited to not more than those eliciting submaximal contraction and addition to the bath was to be made at least 4 min after its prior administration and repeated washings. It has already been demonstrated that the rat stomach fundus is highiy sensitive to 5-hydroxytryptamine 1141 and hence has long been employed as a test object for bioassay of this amine. According to our earlier investigation 171, this preparation was also induced contraction by pa~ially purified substance P. and this
was confirmed again in the present experiment in which synthetic substance P [9] at the concentration of 4-5 x lo-!’ M elicited half maximal contraction of the muscle, a finding indicating a little less efIicacy of substance P to the fundus than neurotensin. It is interesting to note here that xenopsin, an o~tapeptide isolated from frog skin having ~e~rotensin like structure, pyrGlu-Gly-Lys”Arg-pro-Tyr-Ile-Leu~OH), atso showed a rat stomach fundus stimulating activity [I I. Earlier authors reported 1.5,6, 1I] that an isolated guinea pig ileum was also sensitive to neurotensin and contracts. This was confirmed in the present study. The minima1 effective concentration and the mean concentration required to cause the half maximal contraction was 5x 1W”’ M and 1.5x IO-x M, respectively. C-terminal sequences down to the hexapeptide all showed similar activities of one fifth to one tenth that of neurotensin. The tetra- and dipeptide did not have any appreciable activity at concentrations of up to 5~ IO-” M, indicating again that the Arg or Arg-Arg residue is essential for the smooth muscle contraction. The pyrGlu residue at the N-terminal is also essential for the full activity of neurotensin on this test object, a contrast with the stomach fundus. Segments of rat uterus, regardless of their hormonal stage, responded to neurotensin, but their sensitivities were variable and much less than those of rat stomach fundus and the guinea pig ileum, so that they were not studied in detail. In contrast and confirming the earlier reports [ 51, rat duodenum showed relaxation to neurotensin , but the extent of this relaxation was slight and variable, and hence dose-response relation was unclear. It can be said, therefore, that the uterus or duodenum is not a suitable tissue for neurote~sin bioassay. All these in vitro preparations showed prompt and intense ta~hyphylax~s and unaffectiveness by atrapine. tryptamine and pitibenzamine, similar findings to the rat stomach fundus. Intravenous injection of IO0 nmoles of neurotensin induced a considerable fall in the blood pressure of the rabbit for OS-2 min with practically no aiteration in the heart rate. The effect of the dodecapeptide or shorter peptides were markedly weak on the blood pressure, suggesting that the full length of the molecule is needed to elicit the activity. Desensitization was also marked: however, successive application was possible after a considerable intervals of more than 20 min. Recent rapid progress in neuropeptide research is Iargely due to the development of methods using antiserum to a hormone in question, and in most research hormones are
BIOLOGICAL
ACTIVITY
OF NEUROTENSIN
FRAGMENTS
analysed by simple radioimmunoassay or receptor binding assay. However, localization of active center for antibody binding, receptor binding or pharmacological activity is not always identical and consequently it can be possible that values obtained by different procedures differ significantly. This leads to the necessity of parallel different assays includ-
ing tedious bioassay for certain cases of peptide hormone research. In this connexion, the rat stomach fundus may be a good tool for in vitro bioassay of neurotensin, although elimination of interaction by the fundus active contaminants in brain tissue extract such as S-hydroxytryptamine or substance P should be carefully made in the assay system.
REFERENCES Tachibana, M. Uchiyama, T. Nakajima and T. Yasuhara. Isolation and structure of a new active peptide “Xenopsin” on the smooth muscle, especially on a strip of fun-
8. Kobayashi, R. M., M. Brown and W. Vale. Regional distribution of neurotensin and somatostatin in rat brain. Bruin Res.
dus from a rat stomach, from the skin ofXenopus laevis. Chem. Pharmac. Bull. 21: 2801-2804, 1973. Bissette, Cl., C. B. Nemeroff, P. T. Loosen, A. J. Prange, Jr. and M. A. Lipton. Hypothermia and intolerance to cold induced by intracistemal administration of the hypothalamic peptide neurotensin. Nature (Land.) 262: 607-609, 1976. Carraway, R. and S. E. Leeman. The isolation of a new hypothalamic peptide, neurotensin, from bovine hypothalami. J. hiol. Chem. 248: 6854-6861, 1973. Carraway, R. and S. E. Leeman. The amino acid sequence of a hypothalamic peptide, neurotensin. J. hiol. Chem. 250: 19071911, 1975. Carraway, R. and S. E. Leeman. The synthesis of neurotensin. J. biol. Chem. 250: 1912-1918, 1975. Carraway, R. and S. E. Leeman. Structural requirements for the biological activity of neurotensin, a new vasoactive peptide. In: Peptides: Chemistry, Structure und Biology, edited by R. Walter and J. Meinhofer. Ann Arbor: Ann Arbor Science Publishers, 1975, pp. 679-685. Kataoka, K. Subcellular distribution of 5-hydroxytryptamine in the rabbit brain. Jap. J. Physiol. 12: 623-638, 1%2.
Substance P. Life SC;. 15: 2033-2044, 1974. 10. Prange, A. J., Jr., G. R. Breese, G. D. Jahnke, B. R. Martin, B. R. Cooper, J. M. Cott, I. C. Wilson, L. B. Alltop, M. A. Lipton, G. Bissette, C. B. Nemeroff and P. T. Loosen. Modification of pentobarbital effects by natural and synthetic polypeptides: dissociation of brain and pituitary effect. Life Sci. 16: 1907-1914,
1. Araki, K., S.
2.
3.
4.
5. 6.
7.
126: 58&588, 1977. 9. Leeman, S. E. and E. A. Mroz. Minireview.
1975.
11. Segawa, T., M. Hosokawa, K. Kitagawa and H. Yajima. Contractile activity of synthetic neurotensin and related polypeptides on guinea pig ileum. J. Pharm. Pharmuc. 29: 57-58, 1977. 12 Uhl, G. R. and S. H. Snyder. Regional and subcellular distribution of brain neurotensin. Life Sci. 19: 1827-1832, 1976. 13 Uhl, G. R., J. P. Bennett, Jr. and S. H. Snyder. Neurotensin, a central nervous system peptide: apparent receptor binding in brain membranes. Brcrin Res. 130: 299-313, 1977. 14. Vane, J. R. A sensitive method for the assay of 5-hydroxytryptamine. Br. J. Phrrrmtrc. 12: 344-349, 1957. 15. Yajima, H., K. Kitagawa, T. Segawa, M. Nakano and K. Kataoka. Studies on peptides. Application of trifluoromethanesulphonic acid as a deblocking reagent to the synthesis of neurotensin. Chem. pharmcrc~. Bull. 23: 3299-3301, 1975.
Note Added in Proof
After submitting the manuscript, a report by A. Rokaeus et al., (Actu phurmac. tax. 41: 141-147, 1!377) appeared, demonstrating findings similar to the present results; that neurotensin has a potent stimulating activity on isolated rat stomach fundus.