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Primary structure and pharmacological activity of a nonapeptide related to neuromedin U isolated from chicken intestine
Peptides, Vol. 12, pp. 809-812. ©Pergamon Press plc, 1991. Printed in the U.S.A.
0196-9781/91 $3.00 + .00
Primary Structure and Pharmacological Activity of a Nonapeptide Related to Neuromedin U Isolated From Chicken Intestine F I N B A R R O ' H A R T E , C H A R L E S S. B O C K M A N , * W A N Y U N Z E N G , * P E T E R W. A B E L , * S T E V E N H A R V E Y t A N D J. M I C H A E L C O N L O N 1
Regulatory Peptide Center, Department of Biomedical Sciences and *Department of Pharmacology Creighton University School of Medicine, Omaha, NE 68178 ~Department of Physiology, University of Alberta, Edmonton T6G 2H7 Canada R e c e i v e d 14 M a r c h 1991 O'HARTE, F., C. S. BOCKMAN, W. ZENG, P. W. ABEL, S. HARVEY AND J. M. CONLON. Primary structure and pharmacological activity of a nonapeptide related to neuromedin U isolated from chicken intestine. PEPTIDES 12(4) 809-812, 1991.--An extract of chicken intestine contained neuromedin U-like immunoreactivity (36 pmol/g wet tissue weight). The primary structure of the predominant molecular form (NMU-9), comprising 94% of the total immunoreactivity, was established as: GlyTyr-Phe-Phe-Phe-Arg-Pro-Arg-Asn-NH 2. This sequence differs from that of pig neuromedin U-8 (NMU-8) by the substitution of Leu 3 by Phe and, like the corresponding peptide from the guinea pig, is extended from the NH2-terminus by a Gly residue. A minor component of neuromedin U comprised 25 amino acid residues. An extract of chicken whole brain contained much less NMU-like immunoreactivity (1.5 pmol/g) and the nonapeptide was the only molecular form detected. Synthetic chicken NMU-9 produced a concentration-dependent contraction of smooth muscle from the rat uterus and its effect was unchanged in the presence of tetrodotoxin, atropine and indomethacin. The potency of chicken NMU-9 (ECso 360 ___60 nM; mean---S.E., n = 6) was approximately 8-fold less than that of pig NMU-8 (ECso 46--+8 nM) but the maximum contraction produced by both agonists was not significantly different. Neuromedin U
Chicken
Intestine
Rat (uterus)
NEUROMEDIN U (NMU) is a neuropeptide that was first isolated from pig spinal cord on the basis of its ability to stimulate rat uterine smooth muscle (9). Subsequent studies have shown that NMU is localized to populations of myenteric and submucous plexus neurons in the mammalian small intestine (1,5) and the peptide has been isolated from extracts of gut tissue from the rat (3,8), dog (11), rabbit (7) and guinea pig (10). The spectrum of biological activities of neuromedin U has recently been reviewed (7,11). In the rat and rabbit intestine, NMU was isolated in single molecular forms with 23 and 25 amino acid residues, respectively, but in the pig and dog intestine the biologically active COOH-terminal octapeptide of NMU (NMU-8) was also identified. NMU-8 extended from its NH2-terminus by a glycine residue was isolated from the guinea pig intestine. The occurrence and properties of neuromedin U in lower vertebrates have not been studied extensively. The peptide was isolated in a single molecular form with 25 amino acid residues from the gastrointestinal tract of the frog, Rana temporaria, and was shown to display similar biological activities to its mammalian homologs (4). The present study extends our knowledge of the molecular and functional evolution of this peptide by describing the structural and pharmacological characterization of NMU
from a bird, the domestic chicken. METHOD Chicken NMU-9 was synthesized by Multiple Peptide Systems and purified to apparent homogeneity by reversed-phase HPLC. The identity of the peptide was confu'med by amino acid analysis and Edman degradation. Pig NMU-8 and NMU-25 were supplied by Peninsula Laboratories, Inc. and other reagents by Sigma Chemical Co.
Radioimmunoassay Neuromedin U-like immunoreactivity was measured using an antiserum directed against the COOH-terminus of porcine NMU-8 (3). Concentrations are expressed relative to a synthetic pig NMU-25 standard.
Tissue Extraction The intestine (300 g) from eight fasted white Leghorn chickens was homogenized at 4°C with ethanol/0.7 M HC1 (3:1 v/v,
~Requests for reprints should be addressed to Dr. J. M. Conlon, Department of Biomedical Sciences, Creighton University School of Medicine, Omaha, NE 68178.
809
810 2400 ml) as previously described (11). After centrifugation (1600 x g for 30 min), ethanol was removed from the supernatant under reduced pressure. Peptides were isolated from the solution using ten Sep-Pak C-18 cartridges connected in series (11). Bound material was eluted with 70% (v/v) acetonitrile/water and lyophilized. Whole brain (361 g) from approximately 400 chickens was extracted by the same procedure used for the intestinal tissue.
O'HARTE ET AL.
1
i°I/
2OOO
50
7 ~
Purification of Chicken NMU-9 I1
The extract of chicken gut, after partial purification on SepPak cartridges, was redissolved in 0.1% tfifluoroacetic acid/water (10 ml) and injected onto a (250x 10 mm) Supelcosil LC18-DB column (Supelco Inc.) equilibrated with 0.1% trifluoroacetic acid/water at a flow rate of 2 ml/min. The concentration of acetonitrile in the eluting solvent was raised to 21% (v/v) over 10 min, maintained at this concentration for 65 rain and raised to 49% (v/v) over 60 min using linear gradients. Absorbance was measured at 214 rim. Fractions (1 min) were collected and assayed for NMU-LI at appropriate dilution. The fractions comprising peak I (Fig. 1A) were pooled and injected onto a (250x 10 ram) Vydac 218TP510 (C-18) column (Separations Group) equilibrated with 0.1% trifluoroacetic acid/water at a flow rate of 2 ml/min. The concentration of acetonitrile in the eluting solvent was raised to 17.5% (v/v) over 10 min and to 38.5% over 60 min using linear gradients. The fraction containing NMU-LI (denoted by the bar in Fig. 1B) was rechromatographed on a (250 x 4 . 6 mm) Vydac 214TP54 (C-4) column equilibrated with 0.1% trifluoroacetic acid/water at a flow rate of 1.5 ml/min. The concentration of acetonitrile was raised to 17.5% (v/v) over 10 rain and to 28% (v/v) over 30 min using linear gradients. Chicken NMU-9 was purified to apparent homogeneity by chromatography on a (150x4.6 mm) Vydac 219TP5415 (phenyl) column equilibrated with 0.1% trifluoroacetic acid at a flow rate of 1.5 ml/min. The concentration of acetonitrile was raised to 14% (v/v) over 10 min and to 24.5% (v/v) over 30 min over linear gradients. Peak II NMU-LI (Fig. 1A) was purified to apparent homogeneity by sequential chromatography on Vydac C-18, C-4 and phenyl columns equilibrated with 0.1% (v/v) Izifluoroacetic acid using linear gradients of acetonitrile for elution.
Structural Characterization The primary structures of the peptides were determined by automated Edman degradation. The amino acid composition of NMU-9 was determined by precolumn derivatization with phenylisothiocyanate. The molecular mass of NMU-9 was determined by plasma desorption time-of-flight mass spectrometry. Details of the methods and instrumentation have been provided previously (7,11).
Pharmacological Studies Longitudinal muscle strips from the uterine horns of Wistar rats (weight 150-200 g) (n = 6) were prepared and maintained as previously described (11). Contractions produced by increasing concentrations of chicken NMU-9, pig NMU-8 and acetylcholine were measured isometrically using parallel muscle strips from each aninlal. At the end of the experiment, the tissues were blotted and weighed. In some experiments, tissue strips (n =6) were equilibrated for 45 rain with 0.1 p~l atropine, 1 ~ tetrodotoxin and 10 p,M indomethacin before addition of the agonist. Concentration-response curves were constructed by plotting mg
2O
4O
TIME (mln) FIG. 1. Reversed-phase HPLC on a Supelco LC-18-DB column of an extract of chicken intestine after partial purification on Sep-Pak C-18 cartridges. The fraction designated peak I contains NMU-9 and peak II contains NMU-25. The dashed line shows the concentration of acetonitrile in the eluting solvent.
of tension produced per mg tissue weight against the negative logarithm of the agonist concentration. Linear regression analysis of data points between 20% and 80% of maximal contraction was used to calculate potencies (ECho). Data are expressed as means---S.E, and comparisons are made using a one-way analysis of variance followed by Newman-Keuls test. A value of p<0.05 was accepted as a significant difference between groups. RESULTS
Concentrations of Neuromedin U The initial extract of chicken intestine contained neuromedin U-like immunoreactivity equivalent to 36 pmol/g wet tissue weight. The corresponding concentration in an extract of whole brain was 1.5 pmol/g. The immunoreactivity in serial dilutions of both extracts diminished in parallel with the pig neuromedin U-25 standard in radioimmunoassay.
Purification of the Peptides The elution profile on a semipreparative reversed-phase Supelcosil LC-18-DB column of the chicken gut extract is shown in Fig. 1. NMU-like immunoreactivity was eluted in two peaks: peak I (containing NMU-9) comprised 94% of the total immunoreactivity in the column effluent. After chromatography of the peak I fractions on a semipreparative Vydac C-18 column (Fig. 2), NMU-LI was eluted as a late eluting shoulder of a poorly resolved peak. Further purification was achieved by chromatography on an analytical Vydac C-4 column (Fig. 3) and the NMU-like immtmoreactivity was associated with a single sharp peak. Chicken NMU was purified to apparent homogeneity on an analytical Vydac phenyl column and the poptide was eluted as a sharp symmetrical peak. The final yield of pure peptide was 3.5 nmol, which represents 32% of the immunoreaetivity in the initial extract. Peak H NMU-LI was purified to apparent homogeneity using the same sequence of chromatography steps used to purify NMU-9. The final yield of pure peptide, estimated by radioimmunoassay, was 60 pmol. Chromatography of the extract of chicken brain, after purification on Sep-Pak cartrklges, on a Supelcosil LC-18-DB column under the conditions shown in Fig. 1 led to the elution of NMU-like immunoreactivity as a single peak with the same
CHICKEN NEUROMEDIN U
811
TABLE 1 A U T O M A T E D E D M A N D E G R A D A T I O N O F CHICKEN N E U R O M E D I N U-9 r-
SO i,i
.-I rr
40~
Cycle No.
PTH-Amino Acid
Yield (pmol)
Gly Tyr Phe Phe Phe Arg Pro Arg Ash
439 370 546 549 543 118 303 100 263
Z
Z n"
20~ <
/,
TIME (min) FIG. 2. Reversed-phase HPLC on semipreparative Vydac C-18 column of chicken NMU-9 (peak I from Fig. 1). The fraction denoted by the bar containedNMU-likeimmunoreactivityand was purified further.
elution volume as peak I (NMU-9). No immunoreactivity was detected at elution of peak II (NMU-25).
Structural Characterization Amino acid analysis of chicken NMU-9 indicated the following composition: Asx 1.0, Gly 1.2, Arg 2.1, Pro 0.9, Tyr 0.8, Phe 2.9. The results of automated Edman degradation of the peptide are shown in Table 1. Unambiguous assignment of amino acid phenylthiohydantoins was possible for nine cycles of operation of the sequenator and no trace of the derivative of glycine was observed during cycle 10. The agreement between the amino acid composition and sequence analysis data was good, demonstrating the full sequence of the peptide had been obtained. The primary structure of the peptide was confirmed by mass spectrometry. Chicken NMU-9 showed a strong signal at m/z 1202_+1, corresponding to the protonated molecular ion (MH+), and a weaker signal at m/z 1224_+ I, corresponding to the sodium cationated molecular ion (MNa+). The calculated molecular mass of the peptide (Mr) is 1202. The presence of an o~-amidated COOH-terminal asparagine residue in the peptide was confirmed by chemical synthesis. A mixture of natural chicken NMU-9 (1 nmol) and the ct-amidated form of synthetic
0.2
5O
.... ---"°'"~"
0
20
/'/"
1 2 3 4 5 6 7 8 9
The detection limit for phenylthiohydantoin(PTH) amino acids is 0.5 pmol.
NMU-9 (1 nmol) was eluted from a Vydac C-4 reversed-phase HPLC column, under the elution conditions shown in Fig. 3, as a single sharp peak. The presence of a COOH-terminal ~t-amidated residue in chicken NMU-9 is consistent with the strong reactivity of the peptide with an antiserum directed against the COOH-terminus of pig NMU-8. In view of the very small amount of pure peak II NMU, all the available material was subjected to automated Edman degradation. The following partial sequence was obtained: Tyr-LysVal-Asn-X-Asn-Leu-Gln-Gly-Ala]°-Gly-Gly-Ile-Gln-X-Arg-GlyTyr-Phe-Phe2°-Phe-Arg-Pro-Arg-X. Residues denoted by X could not be assigned unambiguously. The data suggest, however, that NMU-9 probably represents the COOH-terminal region of a 25 amino acid residue minor component.
Pharmacological Activity of Chicken NMU-9 The abilities of chicken NMU-9, pig NMU-8 and acetylcholine to contract rat uterine smooth muscle are compared in Fig. 4. All agonists produced a concentration-dependent increase in tension that was rapidly reversed by washing the tissue. Chicken NMU-9 (ECso 360_+60 nM) was significantly (p<0.01) less potent than pig NMU-8 (ECso 46_+8 nM) and both peptides were more potent (p<0.01) than acetylcholine (ECso 1140_+ 150 nM). The maximum contraction produced by chicken NMU-9 (24---3 mg/mg tissue) was not significantly different from the corresponding parameters for pig NMU-8 (25---2 mg/mg tissue) and acetylcholine (27 -+4 mg/mg tissue). In a separate group of experiments (n = 6), concentration-response studies for chicken NMU-9 were carried out in the presence of a mixture of atropine (0.1 IxM), tetrodotoxin (1 IxM) and indomethacin (10 0,M). The potency of the peptide (ECso 230---70 nM) in the presence of these agents was not significantly different from in their absence (ECso 290___27 nM) and there was no significant difference in the maximum contraction produced.
10 DISCUSSION 10
2O
3O
TIME (rain) FIG. 3. Reversed-phase HPLC on an analytical Vydac C-4 column of chicken NMU-9. The peak denoted by the bar contained NMU-like immunoreactivityand the peptide was purified to apparent homogeneityon a Vydac phenyl column.
The study has described the isolation and characterization of a nonapeptide from chicken intestine whose primary structure is the same as neuromedin U-9 from the guinea pig (10), except for the substitution Phe for Leu at position 3. The chicken and guinea pig peptides contain an additional glycine residue at position 1 in comparison to NMU-8 from the pig (9) and dog (11).
812
O ' H A R T E ET AL.
30
0--0
CHICKEN NMU-9
1 V--.
20
,/÷
o
I
n
,,,t,
/I/~
,z,, "5
•
o
Z~- ' ' A
O---O~O
I -9
I -8
I -7
I
-6
I
-5
-4
Log M [AGONIST] FIG. 4. Effects of synthetic chicken NMU-9, pig NMU-8 and acetylcholine on the contraction of rat uterine strips. Data points show mean value ---S.E. of responses using tissues from six rats. The potencies (ECho) of each agonist were significantly different (p<0.01) from each other but the maximum contractions produced were not significantly different.
The concentration of NMU in the chicken gut (36 pmol/g) and brain (1.5 pmol) is comparable to that found in the corresponding tissues of those mammals yet studied but is much lower than that measured in the gut (659 pmol/g) and brain (203 pmol/g) of an amphibian (4). It was not possible to characterize fully the minor component of NMU-like immunoreactivity in the chicken gut extract but determination of its partial amino acid sequence suggests that it comprises 25 residues and represents an NH 2terminally extended form of NMU-9. Chicken NMU-25 contains the Arg16-Gly ~7 sequence, which presumably functions as a recognition site for a processing enzyme. Proteolytic cleavages at such "monobasic" cleavage sites in precursor forms of neurohormonal peptides are relatively common (2,12). It is of interest, however, that the Arg16-Gly17 site in rabbit NMU-25 does
not function as a cleavage site and NMU-9 was not detected in an extract of rabbit gut (7). An attempt was made to prepare more chicken NMU-25 using a larger amount of starting tissue (1.2 kg). This tissue was obtained from a commercial facility which necessitated an unavoidable delay of at least 30 min between slaughter of the chicken and freezing the intestine. In this second gut extract, the concentration of NMU-like immunoreactivity was similar to that measured in the first extract but surprisingly NMU-9 was the only molecular form that was detected. This result suggests that NMU-25 represents a very labile intermediate in the processing of chicken proneuromedin U. The structure-activity relationship of NMU peptides has not been studied in detail. The significant (eight-fold) reduction in potency of chicken NMU-9 compared with pig NMU-8 in contracting rat uterine smooth muscle suggests that the Phe 3 residue in NMU-8 (Phe 4 in NMU-9) is important in interaction with the receptor. This conclusion is supported by the observation that the analog [Gly3]NMU-8 showed no intrinsic activity using a guinea pig trachea preparation whereas substitutions at positions 1, 4, 5, and 7 retained some activity (6). It is improbable that the presence of a Gly residue at position 1 in chicken NMU-9 contributes to the reduction in potency as modifications to this site, e.g., [pGlul]NMU-8 (11)and [D-Tyrl]NMU-8 (6), result in analogs with increased potency. The lack of effect of tetrodotoxin, an inhibitor of depolarization-induced neurotransmitter release, atropine, a blocker of muscarinic cholinergic transmission, and indomethacin, an inhibitor of eicosanoid biosynthesis, on the activity of chicken NMU-9 is consistent with the hypothesis that the contractile action of the peptide is mediated through a direct interaction with specific receptors on smooth muscle cells. Both in the presence and absence of these agents, the maximum contraction produced by chicken NMU-9 and pig NMU-8 was not significantly different from the maximum contraction produced by acetylcholine. ACKNOWLEDGEMENTS This work was supported by the Nebraska Heart Association. The authors thank Dr. Lars Thim, Novo Nordisk A/S, Bagsvaerd, Denmark for mass spectrometry measurements.
REFERENCES 1. Augood, S. J.; Keast, J. R.; Emson, P. C. Distribution and characterization of neuromedin U-like immunoreactivity in rat brain and intestine and in guinea pig intestine. Regul. Pept. 20:281-292; 1988. 2. Benoit, R.; Ling, N.; Esch, F. A new prosomatostatin-derived peptide reveals a pattern for prohormone cleavage at monobasic sites. Science 238:1126-1129; 1987. 3. Conlon, J. M.; Domin, J.; Thim, L.; Dimarzo, V.; Morris, H. R.; Bloom, S. R. Primary structure of neuromedin U from the rat. J. Neurochem. 51:988-991; 1988. 4. Domin, J.; Yiangou, Y.; Spokes, R. A.; Aitkin, A.; Parmar, K. B.; Chrysanthou, B. J.; Bloom, S. R. The distribution, purification and pharmacological action of an amphibian neuromedin U. J. Biol. Chem. 264:20881-20885; 1989. 5. Furness, J. B.; Pompolo, S.; Murphy, R.; Giraud, A. Projections of neurons with neuromedin U-like immunoreactivity in the small intestine of the guinea pig. Cell Tissue Res. 257:415-427; 1989. 6. Hashimoto, T.; Masui, H.; Sakura, N.; Okimura, K.; Uchida, Y. Agonistic and antagonistic activities of neuromedin U-8 analogs on isolated smooth muscles. In: Rivier, J. E.; Marshall, G. R., eds. Peptides--chemistry, structure and biology. Leiden: ESCOM; 1990:
116-117. 7. Kage, R.; O'Harte, F.; Thim, L.; Conlon, J. M. Rabbit neuromedin U-25: Lack of conservation of a posttranslational processing site. Regul. Pept. 33:191-198; 1991. 8. Minamino, N.; Kangawa, K.; Honzawa, M.; Matsuo, H. Isolation and structural determination of rat neuromedin U. Biochem. Biophys. Res. Commun. 156:355-360; 1988. 9. Minamino, N.; Kangawa, K.; Matsuo, H. Neuromedin U-8 and U-25: Novel uterus stimulating and hypertensive peptides identified in porcine spinal cord. Biochem. Biophys. Res. Commun. 130: 1078-1085; 1985. 10. Murphy, R.; Turner, C. A.; Furness, J. B.; Parker, L.; Giraud, A. Isolation and microsequence analysis of a novel form of neuromedin U from guinea pig small intestine. Peptides 11:613-617; 1990. 11. O'Harte, F.; Bockman, C. S.; Abel, P. W.; Conlon, J. M. Isolation, structural characterization and pharmacological activity of dog neuromedin U. Peptides 12:11-15; 1991. 12. Schwartz, T. W. The processing of peptide precursors. 'Proline-diretted arginyl cleavage' and other monobasic processing mechanisms. FEBS Lett. 200:1-10; 1986.
0196-9781/91 $3.00 + .00
Primary Structure and Pharmacological Activity of a Nonapeptide Related to Neuromedin U Isolated From Chicken Intestine F I N B A R R O ' H A R T E , C H A R L E S S. B O C K M A N , * W A N Y U N Z E N G , * P E T E R W. A B E L , * S T E V E N H A R V E Y t A N D J. M I C H A E L C O N L O N 1
Regulatory Peptide Center, Department of Biomedical Sciences and *Department of Pharmacology Creighton University School of Medicine, Omaha, NE 68178 ~Department of Physiology, University of Alberta, Edmonton T6G 2H7 Canada R e c e i v e d 14 M a r c h 1991 O'HARTE, F., C. S. BOCKMAN, W. ZENG, P. W. ABEL, S. HARVEY AND J. M. CONLON. Primary structure and pharmacological activity of a nonapeptide related to neuromedin U isolated from chicken intestine. PEPTIDES 12(4) 809-812, 1991.--An extract of chicken intestine contained neuromedin U-like immunoreactivity (36 pmol/g wet tissue weight). The primary structure of the predominant molecular form (NMU-9), comprising 94% of the total immunoreactivity, was established as: GlyTyr-Phe-Phe-Phe-Arg-Pro-Arg-Asn-NH 2. This sequence differs from that of pig neuromedin U-8 (NMU-8) by the substitution of Leu 3 by Phe and, like the corresponding peptide from the guinea pig, is extended from the NH2-terminus by a Gly residue. A minor component of neuromedin U comprised 25 amino acid residues. An extract of chicken whole brain contained much less NMU-like immunoreactivity (1.5 pmol/g) and the nonapeptide was the only molecular form detected. Synthetic chicken NMU-9 produced a concentration-dependent contraction of smooth muscle from the rat uterus and its effect was unchanged in the presence of tetrodotoxin, atropine and indomethacin. The potency of chicken NMU-9 (ECso 360 ___60 nM; mean---S.E., n = 6) was approximately 8-fold less than that of pig NMU-8 (ECso 46--+8 nM) but the maximum contraction produced by both agonists was not significantly different. Neuromedin U
Chicken
Intestine
Rat (uterus)
NEUROMEDIN U (NMU) is a neuropeptide that was first isolated from pig spinal cord on the basis of its ability to stimulate rat uterine smooth muscle (9). Subsequent studies have shown that NMU is localized to populations of myenteric and submucous plexus neurons in the mammalian small intestine (1,5) and the peptide has been isolated from extracts of gut tissue from the rat (3,8), dog (11), rabbit (7) and guinea pig (10). The spectrum of biological activities of neuromedin U has recently been reviewed (7,11). In the rat and rabbit intestine, NMU was isolated in single molecular forms with 23 and 25 amino acid residues, respectively, but in the pig and dog intestine the biologically active COOH-terminal octapeptide of NMU (NMU-8) was also identified. NMU-8 extended from its NH2-terminus by a glycine residue was isolated from the guinea pig intestine. The occurrence and properties of neuromedin U in lower vertebrates have not been studied extensively. The peptide was isolated in a single molecular form with 25 amino acid residues from the gastrointestinal tract of the frog, Rana temporaria, and was shown to display similar biological activities to its mammalian homologs (4). The present study extends our knowledge of the molecular and functional evolution of this peptide by describing the structural and pharmacological characterization of NMU
from a bird, the domestic chicken. METHOD Chicken NMU-9 was synthesized by Multiple Peptide Systems and purified to apparent homogeneity by reversed-phase HPLC. The identity of the peptide was confu'med by amino acid analysis and Edman degradation. Pig NMU-8 and NMU-25 were supplied by Peninsula Laboratories, Inc. and other reagents by Sigma Chemical Co.
Radioimmunoassay Neuromedin U-like immunoreactivity was measured using an antiserum directed against the COOH-terminus of porcine NMU-8 (3). Concentrations are expressed relative to a synthetic pig NMU-25 standard.
Tissue Extraction The intestine (300 g) from eight fasted white Leghorn chickens was homogenized at 4°C with ethanol/0.7 M HC1 (3:1 v/v,
~Requests for reprints should be addressed to Dr. J. M. Conlon, Department of Biomedical Sciences, Creighton University School of Medicine, Omaha, NE 68178.
809
810 2400 ml) as previously described (11). After centrifugation (1600 x g for 30 min), ethanol was removed from the supernatant under reduced pressure. Peptides were isolated from the solution using ten Sep-Pak C-18 cartridges connected in series (11). Bound material was eluted with 70% (v/v) acetonitrile/water and lyophilized. Whole brain (361 g) from approximately 400 chickens was extracted by the same procedure used for the intestinal tissue.
O'HARTE ET AL.
1
i°I/
2OOO
50
7 ~
Purification of Chicken NMU-9 I1
The extract of chicken gut, after partial purification on SepPak cartridges, was redissolved in 0.1% tfifluoroacetic acid/water (10 ml) and injected onto a (250x 10 mm) Supelcosil LC18-DB column (Supelco Inc.) equilibrated with 0.1% trifluoroacetic acid/water at a flow rate of 2 ml/min. The concentration of acetonitrile in the eluting solvent was raised to 21% (v/v) over 10 min, maintained at this concentration for 65 rain and raised to 49% (v/v) over 60 min using linear gradients. Absorbance was measured at 214 rim. Fractions (1 min) were collected and assayed for NMU-LI at appropriate dilution. The fractions comprising peak I (Fig. 1A) were pooled and injected onto a (250x 10 ram) Vydac 218TP510 (C-18) column (Separations Group) equilibrated with 0.1% trifluoroacetic acid/water at a flow rate of 2 ml/min. The concentration of acetonitrile in the eluting solvent was raised to 17.5% (v/v) over 10 min and to 38.5% over 60 min using linear gradients. The fraction containing NMU-LI (denoted by the bar in Fig. 1B) was rechromatographed on a (250 x 4 . 6 mm) Vydac 214TP54 (C-4) column equilibrated with 0.1% trifluoroacetic acid/water at a flow rate of 1.5 ml/min. The concentration of acetonitrile was raised to 17.5% (v/v) over 10 rain and to 28% (v/v) over 30 min using linear gradients. Chicken NMU-9 was purified to apparent homogeneity by chromatography on a (150x4.6 mm) Vydac 219TP5415 (phenyl) column equilibrated with 0.1% trifluoroacetic acid at a flow rate of 1.5 ml/min. The concentration of acetonitrile was raised to 14% (v/v) over 10 min and to 24.5% (v/v) over 30 min over linear gradients. Peak II NMU-LI (Fig. 1A) was purified to apparent homogeneity by sequential chromatography on Vydac C-18, C-4 and phenyl columns equilibrated with 0.1% (v/v) Izifluoroacetic acid using linear gradients of acetonitrile for elution.
Structural Characterization The primary structures of the peptides were determined by automated Edman degradation. The amino acid composition of NMU-9 was determined by precolumn derivatization with phenylisothiocyanate. The molecular mass of NMU-9 was determined by plasma desorption time-of-flight mass spectrometry. Details of the methods and instrumentation have been provided previously (7,11).
Pharmacological Studies Longitudinal muscle strips from the uterine horns of Wistar rats (weight 150-200 g) (n = 6) were prepared and maintained as previously described (11). Contractions produced by increasing concentrations of chicken NMU-9, pig NMU-8 and acetylcholine were measured isometrically using parallel muscle strips from each aninlal. At the end of the experiment, the tissues were blotted and weighed. In some experiments, tissue strips (n =6) were equilibrated for 45 rain with 0.1 p~l atropine, 1 ~ tetrodotoxin and 10 p,M indomethacin before addition of the agonist. Concentration-response curves were constructed by plotting mg
2O
4O
TIME (mln) FIG. 1. Reversed-phase HPLC on a Supelco LC-18-DB column of an extract of chicken intestine after partial purification on Sep-Pak C-18 cartridges. The fraction designated peak I contains NMU-9 and peak II contains NMU-25. The dashed line shows the concentration of acetonitrile in the eluting solvent.
of tension produced per mg tissue weight against the negative logarithm of the agonist concentration. Linear regression analysis of data points between 20% and 80% of maximal contraction was used to calculate potencies (ECho). Data are expressed as means---S.E, and comparisons are made using a one-way analysis of variance followed by Newman-Keuls test. A value of p<0.05 was accepted as a significant difference between groups. RESULTS
Concentrations of Neuromedin U The initial extract of chicken intestine contained neuromedin U-like immunoreactivity equivalent to 36 pmol/g wet tissue weight. The corresponding concentration in an extract of whole brain was 1.5 pmol/g. The immunoreactivity in serial dilutions of both extracts diminished in parallel with the pig neuromedin U-25 standard in radioimmunoassay.
Purification of the Peptides The elution profile on a semipreparative reversed-phase Supelcosil LC-18-DB column of the chicken gut extract is shown in Fig. 1. NMU-like immunoreactivity was eluted in two peaks: peak I (containing NMU-9) comprised 94% of the total immunoreactivity in the column effluent. After chromatography of the peak I fractions on a semipreparative Vydac C-18 column (Fig. 2), NMU-LI was eluted as a late eluting shoulder of a poorly resolved peak. Further purification was achieved by chromatography on an analytical Vydac C-4 column (Fig. 3) and the NMU-like immtmoreactivity was associated with a single sharp peak. Chicken NMU was purified to apparent homogeneity on an analytical Vydac phenyl column and the poptide was eluted as a sharp symmetrical peak. The final yield of pure peptide was 3.5 nmol, which represents 32% of the immunoreaetivity in the initial extract. Peak H NMU-LI was purified to apparent homogeneity using the same sequence of chromatography steps used to purify NMU-9. The final yield of pure peptide, estimated by radioimmunoassay, was 60 pmol. Chromatography of the extract of chicken brain, after purification on Sep-Pak cartrklges, on a Supelcosil LC-18-DB column under the conditions shown in Fig. 1 led to the elution of NMU-like immunoreactivity as a single peak with the same
CHICKEN NEUROMEDIN U
811
TABLE 1 A U T O M A T E D E D M A N D E G R A D A T I O N O F CHICKEN N E U R O M E D I N U-9 r-
SO i,i
.-I rr
40~
Cycle No.
PTH-Amino Acid
Yield (pmol)
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TIME (min) FIG. 2. Reversed-phase HPLC on semipreparative Vydac C-18 column of chicken NMU-9 (peak I from Fig. 1). The fraction denoted by the bar containedNMU-likeimmunoreactivityand was purified further.
elution volume as peak I (NMU-9). No immunoreactivity was detected at elution of peak II (NMU-25).
Structural Characterization Amino acid analysis of chicken NMU-9 indicated the following composition: Asx 1.0, Gly 1.2, Arg 2.1, Pro 0.9, Tyr 0.8, Phe 2.9. The results of automated Edman degradation of the peptide are shown in Table 1. Unambiguous assignment of amino acid phenylthiohydantoins was possible for nine cycles of operation of the sequenator and no trace of the derivative of glycine was observed during cycle 10. The agreement between the amino acid composition and sequence analysis data was good, demonstrating the full sequence of the peptide had been obtained. The primary structure of the peptide was confirmed by mass spectrometry. Chicken NMU-9 showed a strong signal at m/z 1202_+1, corresponding to the protonated molecular ion (MH+), and a weaker signal at m/z 1224_+ I, corresponding to the sodium cationated molecular ion (MNa+). The calculated molecular mass of the peptide (Mr) is 1202. The presence of an o~-amidated COOH-terminal asparagine residue in the peptide was confirmed by chemical synthesis. A mixture of natural chicken NMU-9 (1 nmol) and the ct-amidated form of synthetic
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The detection limit for phenylthiohydantoin(PTH) amino acids is 0.5 pmol.
NMU-9 (1 nmol) was eluted from a Vydac C-4 reversed-phase HPLC column, under the elution conditions shown in Fig. 3, as a single sharp peak. The presence of a COOH-terminal ~t-amidated residue in chicken NMU-9 is consistent with the strong reactivity of the peptide with an antiserum directed against the COOH-terminus of pig NMU-8. In view of the very small amount of pure peak II NMU, all the available material was subjected to automated Edman degradation. The following partial sequence was obtained: Tyr-LysVal-Asn-X-Asn-Leu-Gln-Gly-Ala]°-Gly-Gly-Ile-Gln-X-Arg-GlyTyr-Phe-Phe2°-Phe-Arg-Pro-Arg-X. Residues denoted by X could not be assigned unambiguously. The data suggest, however, that NMU-9 probably represents the COOH-terminal region of a 25 amino acid residue minor component.
Pharmacological Activity of Chicken NMU-9 The abilities of chicken NMU-9, pig NMU-8 and acetylcholine to contract rat uterine smooth muscle are compared in Fig. 4. All agonists produced a concentration-dependent increase in tension that was rapidly reversed by washing the tissue. Chicken NMU-9 (ECso 360_+60 nM) was significantly (p<0.01) less potent than pig NMU-8 (ECso 46_+8 nM) and both peptides were more potent (p<0.01) than acetylcholine (ECso 1140_+ 150 nM). The maximum contraction produced by chicken NMU-9 (24---3 mg/mg tissue) was not significantly different from the corresponding parameters for pig NMU-8 (25---2 mg/mg tissue) and acetylcholine (27 -+4 mg/mg tissue). In a separate group of experiments (n = 6), concentration-response studies for chicken NMU-9 were carried out in the presence of a mixture of atropine (0.1 IxM), tetrodotoxin (1 IxM) and indomethacin (10 0,M). The potency of the peptide (ECso 230---70 nM) in the presence of these agents was not significantly different from in their absence (ECso 290___27 nM) and there was no significant difference in the maximum contraction produced.
10 DISCUSSION 10
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TIME (rain) FIG. 3. Reversed-phase HPLC on an analytical Vydac C-4 column of chicken NMU-9. The peak denoted by the bar contained NMU-like immunoreactivityand the peptide was purified to apparent homogeneityon a Vydac phenyl column.
The study has described the isolation and characterization of a nonapeptide from chicken intestine whose primary structure is the same as neuromedin U-9 from the guinea pig (10), except for the substitution Phe for Leu at position 3. The chicken and guinea pig peptides contain an additional glycine residue at position 1 in comparison to NMU-8 from the pig (9) and dog (11).
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Log M [AGONIST] FIG. 4. Effects of synthetic chicken NMU-9, pig NMU-8 and acetylcholine on the contraction of rat uterine strips. Data points show mean value ---S.E. of responses using tissues from six rats. The potencies (ECho) of each agonist were significantly different (p<0.01) from each other but the maximum contractions produced were not significantly different.
The concentration of NMU in the chicken gut (36 pmol/g) and brain (1.5 pmol) is comparable to that found in the corresponding tissues of those mammals yet studied but is much lower than that measured in the gut (659 pmol/g) and brain (203 pmol/g) of an amphibian (4). It was not possible to characterize fully the minor component of NMU-like immunoreactivity in the chicken gut extract but determination of its partial amino acid sequence suggests that it comprises 25 residues and represents an NH 2terminally extended form of NMU-9. Chicken NMU-25 contains the Arg16-Gly ~7 sequence, which presumably functions as a recognition site for a processing enzyme. Proteolytic cleavages at such "monobasic" cleavage sites in precursor forms of neurohormonal peptides are relatively common (2,12). It is of interest, however, that the Arg16-Gly17 site in rabbit NMU-25 does
not function as a cleavage site and NMU-9 was not detected in an extract of rabbit gut (7). An attempt was made to prepare more chicken NMU-25 using a larger amount of starting tissue (1.2 kg). This tissue was obtained from a commercial facility which necessitated an unavoidable delay of at least 30 min between slaughter of the chicken and freezing the intestine. In this second gut extract, the concentration of NMU-like immunoreactivity was similar to that measured in the first extract but surprisingly NMU-9 was the only molecular form that was detected. This result suggests that NMU-25 represents a very labile intermediate in the processing of chicken proneuromedin U. The structure-activity relationship of NMU peptides has not been studied in detail. The significant (eight-fold) reduction in potency of chicken NMU-9 compared with pig NMU-8 in contracting rat uterine smooth muscle suggests that the Phe 3 residue in NMU-8 (Phe 4 in NMU-9) is important in interaction with the receptor. This conclusion is supported by the observation that the analog [Gly3]NMU-8 showed no intrinsic activity using a guinea pig trachea preparation whereas substitutions at positions 1, 4, 5, and 7 retained some activity (6). It is improbable that the presence of a Gly residue at position 1 in chicken NMU-9 contributes to the reduction in potency as modifications to this site, e.g., [pGlul]NMU-8 (11)and [D-Tyrl]NMU-8 (6), result in analogs with increased potency. The lack of effect of tetrodotoxin, an inhibitor of depolarization-induced neurotransmitter release, atropine, a blocker of muscarinic cholinergic transmission, and indomethacin, an inhibitor of eicosanoid biosynthesis, on the activity of chicken NMU-9 is consistent with the hypothesis that the contractile action of the peptide is mediated through a direct interaction with specific receptors on smooth muscle cells. Both in the presence and absence of these agents, the maximum contraction produced by chicken NMU-9 and pig NMU-8 was not significantly different from the maximum contraction produced by acetylcholine. ACKNOWLEDGEMENTS This work was supported by the Nebraska Heart Association. The authors thank Dr. Lars Thim, Novo Nordisk A/S, Bagsvaerd, Denmark for mass spectrometry measurements.
REFERENCES 1. Augood, S. J.; Keast, J. R.; Emson, P. C. Distribution and characterization of neuromedin U-like immunoreactivity in rat brain and intestine and in guinea pig intestine. Regul. Pept. 20:281-292; 1988. 2. Benoit, R.; Ling, N.; Esch, F. A new prosomatostatin-derived peptide reveals a pattern for prohormone cleavage at monobasic sites. Science 238:1126-1129; 1987. 3. Conlon, J. M.; Domin, J.; Thim, L.; Dimarzo, V.; Morris, H. R.; Bloom, S. R. Primary structure of neuromedin U from the rat. J. Neurochem. 51:988-991; 1988. 4. Domin, J.; Yiangou, Y.; Spokes, R. A.; Aitkin, A.; Parmar, K. B.; Chrysanthou, B. J.; Bloom, S. R. The distribution, purification and pharmacological action of an amphibian neuromedin U. J. Biol. Chem. 264:20881-20885; 1989. 5. Furness, J. B.; Pompolo, S.; Murphy, R.; Giraud, A. Projections of neurons with neuromedin U-like immunoreactivity in the small intestine of the guinea pig. Cell Tissue Res. 257:415-427; 1989. 6. Hashimoto, T.; Masui, H.; Sakura, N.; Okimura, K.; Uchida, Y. Agonistic and antagonistic activities of neuromedin U-8 analogs on isolated smooth muscles. In: Rivier, J. E.; Marshall, G. R., eds. Peptides--chemistry, structure and biology. Leiden: ESCOM; 1990:
116-117. 7. Kage, R.; O'Harte, F.; Thim, L.; Conlon, J. M. Rabbit neuromedin U-25: Lack of conservation of a posttranslational processing site. Regul. Pept. 33:191-198; 1991. 8. Minamino, N.; Kangawa, K.; Honzawa, M.; Matsuo, H. Isolation and structural determination of rat neuromedin U. Biochem. Biophys. Res. Commun. 156:355-360; 1988. 9. Minamino, N.; Kangawa, K.; Matsuo, H. Neuromedin U-8 and U-25: Novel uterus stimulating and hypertensive peptides identified in porcine spinal cord. Biochem. Biophys. Res. Commun. 130: 1078-1085; 1985. 10. Murphy, R.; Turner, C. A.; Furness, J. B.; Parker, L.; Giraud, A. Isolation and microsequence analysis of a novel form of neuromedin U from guinea pig small intestine. Peptides 11:613-617; 1990. 11. O'Harte, F.; Bockman, C. S.; Abel, P. W.; Conlon, J. M. Isolation, structural characterization and pharmacological activity of dog neuromedin U. Peptides 12:11-15; 1991. 12. Schwartz, T. W. The processing of peptide precursors. 'Proline-diretted arginyl cleavage' and other monobasic processing mechanisms. FEBS Lett. 200:1-10; 1986.