To develop a highly potent agonist and to examine the structure-contractile activity relationship of neuromedin U-8(NMU-8), seventeen analogs were synthesized and tested for contractile activity on isolated chicken crop smooth muscle preparations. The analogs were designed to examine the contributions of cyclic structure and acidic function at the N-terminal of NMU-8 and NMU-8(2--8) to the biological activity. The relative activity (RA) values of NMU-8 analogs were as follows: [Dp-Glu1]-NMU-8,5.50; [pyrohomoglutamyl(pHgu)1]-NMU-8,4.65;[D-pHgu1]-NMU-8, 4.66; [Asp1]-NMU-8, 11.4; [acetyl(Ac)-Asp1]-NMU-8, 9.81; [Ac-Glu1]- NMU-8, 18.6; [succinyl (Suc)-Tyr1-NMU-8,69.3; [3-sulfoalanyl (Sal)1]-NMU-8, 12.7. The RA values of NMU-8(2--8) analogs were as follows: alpha-picolinyl (Pic)-NMU-8 (2--8), 7.96; 2-furoyl (Fur)-NMU-8, (2--8), 9.91; 2-thiophenecarboxyl (Thi)-NMU-8 (2--8), 3.41; 4-hydroxyphenylpropionyl (Hpp)-NMU-8(2--8), 3.20; o-phthalyl (Pht)-NMU-8 (2--8), 11.3; Suc-NMU-8 (2--8), 109; malonyl (Mlo)-NMU-8 (2--8), 17.9; maleyl (Mle)-NMU-8 (2--8), 31.6; glutaryl (Glt)-NMU-8 (2--8), 81.3; The potencies of the analogs were higher than that of p-NMU-8. Suc-NMU-8 (2--8) showed the highest potency among the analogs synthesized. The results reveal that the carboxylic acid group at the N-terminus of NMU-8 makes a major contribution to the activity.
Rat neuromedin U (r-NMU) and its fragment peptide amides were synthesized by solid-phase methodology. Using a chicken crop smooth muscle contraction assay, the potency of r-NMU and its fragments relative to porcine neuromedin U-8 (p-NMU-8) was r-NMU: 10.25 +/- 2.88, r-NMU (6-23): 8.01 +/- 1.04, r-NMU (10-23): 2.76 +/- 0.46, r-NMU (13-23): 2.81 +/- 0.52, and r-NMU (16-23): 0.88 +/- 0.19, respectively. Two heptapeptides, r-NMU (17-23) and r-NMU (16-22), had a relative potency of 0.61 and 0.03 respectively, and elicited maximal contraction at a dose of 10 microM to a similar degree to p-NMU-8. The other shorter C-terminal fragments did not elicit the maximal contraction or any activity. In a rat uterus contraction assay, r-NMU (13-23), but not r-NMU (16-23), at a dose of 4 nM retained as high a stimulatory activity as r-NMU itself. r-NMU (17-22) was the smallest peptide fragment to elicit the maximal sustained contraction at 10 microM. These results indicate that the amino acid sequence Phe-Leu-Phe-Arg-Pro-Arg, corresponding to positions 17 to 22 of r-NMU, may be essential for contractile activity. N-terminal peptide segments Tyr-Gln-Gly-Pro corresponding to positions 6 to 9, and Ser-Gly-Gly corresponding to positions 13 to 15, appear to be of special importance for potent activity.
Contractile activity of porcine neuromedin U-25 (p-NMU-25) and various neuromedin U (NMU) peptide fragment amides was examined on chicken crop smooth muscle preparation. The relative activity (expressed as RA value) of p-NMU-25 to porcine neuromedin U-8 (p-NMU-8) was 5.51 +/- 0.09, and p-NMU-25 (15-25) was the most potent fragment with an RA value of 7.78 +/- 0.05. All C-terminal 11-peptide amides of rat, rabbit, and frog NMU peptides retained activity about three-fold higher than the corresponding C-terminal 8-peptide amides. The peptide segment Asn15-Arg-Arg17 of p-NMU-25, as well as the corresponding positions of various NMU peptides: Ser13-Gly-Gly15 of rat NMU and Ser15-Arg-Gly17 of rabbit and frog NMUs, appeared to be involved in the structural requirements for increased contractile activity in the assay system.
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