KR100664998B1 - Neuropeptide Having Relaxing Activity of Smooth Muscle - Google Patents

Abstract

The present invention relates to a new neuropeptide having a relaxing activity isolated from the dermal animal, and more particularly to a new neuropeptide showing a smooth muscle relaxation activity having the amino acid sequence of SEQ ID NO: 2.

The peptide of the present invention is expected to be useful as a muscle relaxant because it exhibits a stronger relaxation activity than the previously known relaxation activity peptides S1 and S2, but is composed of a small number of amino acids and is easy to mass produce.

Echinoderm, Starfish (Asterina pectinifera), Neuropeptides, Relaxing activity

Description

Neuropeptide Having Relaxing Activity of Smooth Muscle

Figure 1 shows the results of the measurement of the relaxation activity for the starfish DRM (dorsal retractor muscle) of the four fractions separated from the starfish using a Sep-Pak C18 column.

Figure 2 shows the relaxation activity for CM52 cation exchange resin chromatogram and starfish DRM (■ indicates the active fraction).

Figure 3 shows the relaxation activity against reverse phase HPLC chromatogram and starfish DRM of the active fraction of Figure 2 (■ represents the active fraction).

Figure 4 shows the relaxation activity for the anion exchange resin chromatogram and starfish DRM of the active fraction of Figure 3 (■ represents the active fraction).

Figure 5 shows the relaxation activity against reverse phase HPLC chromatogram and starfish DRM of the active fraction of Figure 4 (■ indicates the active fraction).

Figure 6 shows the relaxation activity against reverse phase HPLC chromatogram and starfish DRM of the active fraction of Figure 5 (■ represents the active fraction).

FIG. 7 shows MALDI-TOF Mass results of the final purification step (A) of the active fraction of FIG. 6, relaxation activity (B) and activity peaks for starfish DRM.

FIG. 8 is a graph showing concentration-dependent relaxation activity measurement results for starfish DRM of Asteripectin ((), S1 (○), S2 (▼), FLRF (▽), and FMRFa (□).

The present invention relates to a new neuropeptide having a relaxing activity isolated from the dermal animal, and more particularly to a new neuropeptide showing a smooth muscle relaxation activity having the amino acid sequence of SEQ ID NO: 2.

The regulation of smooth muscle contraction and relaxation in the function of neuronal peptides is regulated by the combination of myosina and actin. Myosin light-chain kinase phosphorylates the myosin regulatory light chain, causing myosin to bind to actin, resulting in contractile action, while relaxation of myosin-actin binding occurs. Mammalian smooth muscle, which does not have structural patterns, constitutes the walls of organs and circulatory systems of the intestine, and is known to be involved in the transport, degradation, release, preservation and maintenance of contents.

These smooth muscles have been reported to be present as catch muscle fibers that contain filaments and dense bodies, as well as in ripples in mammals as well as in evolutionary molluscs. For this reason, various studies on the smooth muscle regulation of neuronal peptides using mollusks as well as mammals have been conducted, and mainly the catch relaxing peptides of molluscs. Typically, the separation of FMRFamide with a relaxation activity against acetylcholine stimulation by the acetylcholine stimulation from the pearl ganglia pedal ganglia has been reported, and the small cardioactive peptide and Mytilus which increase the contractile force of ABRM by electrical stimulation from ABRM Inhibitor peptide was purified.

However, muscle physiology of echinoderm, which is recognized as an evolutionary species more closely related to vertebrates than mollusks, is thought to be more similar to that of vertebrates than that of mollusks, but studies using them are insufficient. Recently, extracts from eukaryotic animals have been identified to induce relaxation in vitro.However , studies on neurotransmitters in eukaryotic animals have been conducted using basic neurotransmitters such as acetylcholine, catecholamine and GABA. Limited to active studies. In addition, most of the star starfish used as experimental animals in the present invention are disposed of, and studies on the verification of the active effects on bioactive substances or on the relationship between separation, purification and structural activity have been extremely limited.

Accordingly, the present inventors earnestly try to isolate a new neuropeptide having a relaxing activity against the starfish, a marine organism, and obtain a methanol-acid extract from the starfish, Asterina pectinifera , and extract the CM- After adjustment by cellulose column chromatography, new neuropeptides with relaxation activity were isolated via HPLC system to complete the present invention.

After all, the main object of the present invention is to provide a novel neuronal peptide having smooth muscle relaxation activity.

Another object of the present invention to provide a nucleic acid comprising DNA and RNA encoding the neuronal peptide.

In order to achieve the above object, the present invention provides a neuronal peptide having a smooth muscle relaxation activity having the amino acid sequence of SEQ ID NO: 2.

In the present invention, the peptide may be characterized by having an amino acid sequence of SEQ ID NO: 1, characterized in that the C-terminal is carboxylated or amidated, or the N-terminal amine group is substituted with an alkyl or acetyl group The amino acid sequence may be L-type or D-type.

The present invention also provides a nucleic acid comprising DNA or RNA encoding a peptide of SEQ ID NO: 2, a recombinant vector comprising the nucleic acid and a transformed microorganism obtained by introducing the recombinant vector into bacteria, yeast or fungi.

The present invention also provides a method for producing a neuronal peptide having smooth muscle relaxation activity, characterized in that the culturing the transformed microorganism.

The present invention also provides a pharmaceutical composition for improving any one or more symptoms selected from the group consisting of bronchial asthma, hypertension, diuresis and erectile dysfunction containing the peptide as an active ingredient.

In the present invention, the neuropeptide described in SEQ ID NO: 1 obtains a methanol-acid extract from Asterina pectinifera , which is an dermal animal, and adjusts the extract by CM 52 cation exchange resin column chromatography, followed by HPLC system. Separation was carried out.

The isolated peptide is a novel neuronal peptide having a relaxation activity, and after passing through the HPLC system, a 'MALDI-TOF mass spectrometer (Voyager-DE ^TM STR spectrometer, Perseptive Biosystems, USA)' and 'automated protein sequencer PPSQ-21A ( Shimadzu, Ltd., Japan) 'amino acid sequence was determined (SEQ ID NO: 1).

As a result of measuring the relaxation activity of the starfish with the peptide of SEQ ID NO: 1, the activity was about 10 ³ times higher than S1 and about 10 ⁴ times higher than S2.

In the neurological peptide of the present invention, the C-terminus is preferably carboxylated or aminated, and the N-terminal amine group is preferably substituted with an acetyl group or an alkyl group.

The amino acids constituting the peptide of the present invention can be used any of L-type amino acids and / or D-type amino acids.

Smooth muscle relaxation activity of the peptide of the present invention can be used to alleviate symptoms such as bronchial asthma, hypertension, diuresis and erectile dysfunction associated with muscle contraction and relaxation.

The neurological peptides of the present invention can be obtained by separation and purification from starfish as well as prepared by chemical methods using peptide synthesis, and can be used for microorganisms transformed with recombinant vectors containing DNA or RNA encoding peptides. It may be prepared by culturing.

In the present invention, "vector" refers to a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA in a suitable host. The vector may be a plasmid, phage particles, or simply a potential genomic insert. Once transformed into the appropriate host, the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself. Since plasmids are the most commonly used form of current vectors, "plasmid" and "vector" are sometimes used interchangeably in the context of the present invention. For the purposes of the present invention, it is preferred to use plasmid vectors. Typical plasmid vectors that can be used for this purpose include (a) a replication initiation point that allows for efficient replication to include hundreds of plasmid vectors per host cell, and (b) host cells transformed with the plasmid vector. It has a structure comprising an antibiotic resistance gene and (c) a restriction enzyme cleavage site into which foreign DNA fragments can be inserted. Although no appropriate restriction enzyme cleavage site is present, the use of synthetic oligonucleotide adapters or linkers according to conventional methods facilitates ligation of the vector and foreign DNA.

After ligation, the vector should be transformed into the appropriate host cell. In the present invention, preferred host cells are prokaryotic cells. Suitable prokaryotic host cells include E. coli DH5α, E. col JM101, E. coli K12, E. coli W3110, E. coli X1776, E. coli XL-1Blue (Stratagene), E. coli B, E. coli B21, and the like. It includes. However, E. coli strains such as FMB101, NM522, NM538 and NM539 and other prokaryotic species and genera may also be used. In addition to the aforementioned E. coli , strains of the genus Agrobacterium, such as Agrobacterium A4, bacilli , such as Bacillus subtilis , Salmonella typhimurium or Serratia marghesen another variety of enteric bacteria and Pseudomonas (Pseudomonas) in strains such as marcescens) may be used as host cells.

Prokaryotic transformation can be readily accomplished using the calcium chloride method described in section 1.82 of Sambrook et al., Supra. Alternatively, electroporation (Neumann et al., EMBO J., 1: 841, 1982) can also be used for transformation of these cells.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples only illustrate the present invention in detail and do not limit the present invention by these examples.

Example 1: Extraction of Relaxing Peptide Fractions from Starfish

The starfish used in the present example were starfish ( Asterina pectinifera ), which was collected at Homi Cape in Pohang, Gyeongbuk in August 2002, and 450 starfish of 8 to 15 cm in length were collected and immediately used for extraction.

450 live starfish were placed in a 70% methanol solution and bathed at 65 ° C for 5 minutes. After 5% acetic acid was added to crush the tissue, the supernatant was concentrated by centrifugation (4 ° C., 40 minutes, 10,000 × g), and ethanol was added so that the ratio of sample to ethanol was 2: 9 (v / v). The precipitate was removed by freezing at -20 ° C. and centrifuged (4 ° C., 40 min, 10,000 × g). The separated supernatant was concentrated again and ethanol was added so that the ratio of sample to ethanol was 1:10 (v / v), followed by centrifugation (4 ° C, 40 minutes, 10,000 xg) to remove the precipitate once more. . Supernatant obtained by adding ethanol and NaCl in the ratio of sample (1 L): ethanol (8 L): NaCl (11 g) to this supernatant after centrifugation (4 ° C., 40 minutes, 10,000 × g). After the solution was concentrated, 1 N HCl was added so that the total concentration was 0.1 N HCl, followed by centrifugation (4 ° C., 50 minutes, 20,000 × g).

The supernatant was partially injected into Sep-Pak C ₁₈ cartridge (20 mL, Waters) to separate the material, followed by H ₂ O (DW), 10% methanol (retained materials; RM10) and 60 containing 0.1% TFA. The materials were eluted with% methanol (RM60) and 100% methanol (RM100) to obtain DW fraction, RM10 fraction, RM 60 fraction and RM 100 fraction, respectively.

Example 2. Determination of the Relaxation Activity of Starfish Fraction on Starfish DRM

The relaxation activity of the RM10, RM60, and RM 100 fractions extracted in Example 1 was used by the DRM (dorsal retractor muscle) of Asterina pectinifera .

In order to sample the starfish DRM, the starfish was removed from the eye spot, the DRM across the center was separated with a scalpel, and cut into 20 mm fragments. Each of the prepared fragments was fixed to a support in the reactor, and the upper part was connected to an isometric transducer. After 1 g of tension was applied to the connected tissue, equilibration was performed by replacing artificial seawater (55 mM Mg ²⁺ artificial sea water) every 15 minutes at room temperature. Thereafter, 10 ^-6 M acetylcholine was activated to activate, and after 15 minutes, ^10-6 M acetylcholine was administered to contract the muscles, and the fractions obtained in each HPLC purification step were administered. All procedures were performed under artificial seawater (NaCl 445 mM, KCl 10 mM, CaCl ₂ .2H ₂ O 10 mM, MgCl ₂ .6H ₂ O 55 mM, Glucose 10 mM and Tris-HCl (pH 7.8) 20 mM). As a result, smooth muscle relaxation activity was confirmed in RM60 aliquots (FIG. 1).

Example 3. Isolation and Purification of Neuronal Peptides from RM60 Fractions

Starfish extract RM60 fractions confirmed in Example 2 were separated through seven consecutive HPLC steps using different columns. The fractions obtained through each HPLC purification were measured for the activity of starfish DRM in the same manner as in Example 2, and the fractions showing the activity were concentrated and used for the next step of HPLC purification.

① CM52 column : Concentration gradient using CM52 (2.5 × 30 cm) column, 20 mM → 1.5 M ammonium acetate (pH 5.0, 6 hours); Isolate under flow rate, 2.75 mL / min. Relaxation activity was observed in fractions near 50 min retention time (FIG. 2).

② C ₁₈ column : The concentrated active fraction was applied to a reversed phase HPLC column (Vydac protein & peptide C ₁₈ , 9.2 × 250 mm), containing 0.1% TFA aqueous solution (pH 2.2) as solvent A and 0.1% TFA as solvent B. 100% acetonitrile (actitrile) (pH 2.2) was used to give a concentration gradient from 0 to 100% of solvent B, and the flow rate was separated at 40 ° C at 3.0 mL / min. Relaxation activity was observed in fractions near 50 min retention time (FIG. 3).

③ anion exchange column : purified using TSK-gel DEAE-5PW (7.5 × 75 mm) column, solvent A, 10 mM Tris-HCl buffer (pH 9.2); 10 mM Tris-HCl buffer (pH 9.2) containing B solvent, 1.0 M NaCl; Concentration gradient, 0 → 0.5 MB solvent (100 min), flow rate; 0.5 mL / min, temperature; 40 ° C .; Fraction volume (fraction volume), was carried out under the condition of 1.0 mL. Relaxation activity was observed in fractions near 25 min retention time (FIG. 4).

④ C ₁₈ column : separated using a Capcellpak C ₁₈ (4.6 × 250 mm) column, HPLC was concentration gradient, 15 → 30% B (60 min); Flow rate, 1.0 mL / min; Temperature, was carried out under the conditions of 40 ℃. Relaxation activity was observed in fractions near 35 min retention time (FIG. 5).

⑤ C ₁₈ column : Hypersil-BDS C ₁₈ (2 × 125 mm) was used, which is different from the previous phase reversed column. At this time, the concentration gradient of the solvent, 15 → 30% was separated at 40 ℃ for 60 minutes at a flow rate of 0.5 mL / min for 60 minutes, showed activity in the portion corresponding to 20% acetonitrile (acetonitrile) (Fig. 6).

⑥ Size exclusion column: HPLC was performed using a Superdex Peptide HR 10/30 (10 × 300 mm) column under 30% acetonitrile in order to perform molecular weight separation. At this time, the flow rate was separated at room temperature to 0.5 mL / min, and had a retention time of 24.034 minutes. As a result, it was found that the molecular weight is between about 1000 ~ 3000 Da.

⑦ Hypersil-BDS C ₁₈ column : Finally purified using a Hypersil-BDS C ₁₈ (2 × 125 mm) column under isocratic conditions of 20% acetonitrile. As a result, one pure material having a retention time of 16.1 minutes was finally purified, which was named Asteripectin since it was purified from Asterina pectinifera extract (FIG. 7).

Example 4 Determination of Molecular Weight and Amino Acid Sequence of Natural Peptides

The molecular weight of the peptide Asteripectin having a relaxing activity against the starfish DRM finally purified in Example 3 was measured using a MALDI-TOF mass spectrometer (Voyager-DE ^™ STR spectrometer, Perseptive Biosystems, USA). To determine the amino acid sequence of asteripectin, it was analyzed by Edman digestion using an automated protein sequencer PPSQ-21A (Shimadzu, Ltd., Japan).

The molecular weight of the final purified peptide was 1601.7 Da (FIG. 7) and the amino acid sequence was Phe-Gly-Lys-Gly-Gly-Ala-Tyr-Asp-Pro-Leu-Ser-Ala-Gly-Phe-Thr-Asp It was identified as a composed hexadecapeptide (Table 1).

Amino Acid Sequence of Asteripectin and Mass Spectrometry of Asteripectin Cycles Phenylthiohydantion amino acid Yield (pmol) m / z One Phe 452 1601.72 2 Gly 298 3 Lys 363 4 Gly 258 5 Gly 241 6 Ala 268 7 Tyr 235 8 Asp 143 9 Pro 152 10 Leu 147 11 Ser 26 12 Ala 80 13 Gly 64 14 Phe 80 15 Tyr 23 16 Asp 9

As a result of examining the similarity of the amino acid sequence of Asteripectin, the peptide sequence was identified as a new neuronal peptide having no similarity to the known neuronal peptide.

Neural Peptides Purified from Epidermal Animals Peptide name (species) Sequence References S1 (Asteroidae) 1 5 Gly-Phe-Asn-Ser-Ala-Leu-Met-Phe-amide (SEQ ID NO: 3) Elphick et al. , 1991b, c S2 (Asteroidae) 1 5 10 Ser-Gly-Pro-Tyr-Ser-Phe-Asn-Ser-Gly-Leu-Tyr-Phe-amide (SEQ ID NO: 4) Elphick et al. , 1991b, c GFSKLYFa (Holothuroidea) 1 5 Gly-Phe-Ser-Lys-Leu-Tyr-Phe-amide (SEQ ID NO: 5) Diaz Miranda et al. , 1992 SGTSVLYFa (Holothuroidea) 1 5 Ser-Gly-Tyr-Ser-Val-Leu-Tyr-Phe-amide (SEQ ID NO: 6) Diaz Miranda et al. , 1992 GYSPFMFa (Holothuroidea) 1 5 Ser-Gly-Tyr-Ser-Pro-Phe-Met-Phe-amide (SEQ ID NO: 7) Ohtani et al. , 1999 FKSPFMFa (Holothuroidea) 1 5 Phe-Lys-Ser-Pro-Phe-Met-Phe-amide (SEQ ID NO: 8) Ohtani et al. , 1999

The similarity between the peptide sequence of Asteripectin and known neuronal peptides derived from epidermis was confirmed to be a new neuronal peptide that is completely different from the known peptides.

Example 5: Synthesis and Purification of Peptides

Peptides having the same peptide sequence as that of Asteripectin were synthesized to confirm that they had the same relaxation activity.

Synthesis of Peptides

Purified peptides were synthesized by solid phase method using Fmoc-method according to amino acid sequencing and molecular weight analysis (Park, NG et al ., I. Bull. Korean Chem. Soc. 18:50, 1997.). SF-I was swelled with DMF using Fmoc-Asp (OBut) -resin (0.67 mmole / g) at 0.05 mmole scale and then Fmoc-protected from the resin using 20% piperidine / DMF. Was cut. By using DIPCI / HOBT method, the carboxyl group of the amino acid to be bound is activated in ester form, introduced into the resin, and the completed reaction resin is washed with DMF, and then again with 20% piperidine / DMF. Fmoc-group was cleaved. After the reaction, the mixture was washed with DMF and the second amino acid was bound by DIPCI / HOBT method. This process was repeated sequentially to grow complete peptide chains in the resin.

Cleavage and Deprotection of Peptide-Resins

A cleavage reagent [TFA (4 mL): m -cresol (0.4 mL): thioanisole (0.8 mL): EDT (0.5 mL)] was used to remove the resins and side chain protecting groups linked to the synthetic peptide. The reaction was carried out at room temperature for 90 minutes. After the reaction was completed, the resin was filtered and the peptide solution was concentrated in vacuo. Subsequently, the peptide was crystallized and the cleavage reagent was removed by adding cold ether to the concentrate, dissolved in 5% acetic acid solution, concentrated, and lyophilized.

Purification of Synthetic Peptides

Crude peptides from the resin were treated with Vydac 218TP510 Protein & peptide C ₁₈ (9.2 × 250 mm) to acetonitrile containing 0.1% TFA in a concentration gradient of 18 to 25% with a flow rate of 3.0 mL for 15 minutes. It separated on the condition of / min. Pure synthetic peptides were obtained by isocratic elution of 22% acetonitrile on the same column.

Comparison of natural and synthetic

Purified natural products The identity of the compounds was confirmed by comparing the molecular weight measurements and retention time (RT) using a reverse-phase HPLC column. Purified synthetic peptides were compared under isocratic conditions of 22% acetonitrile using Vydac protein & peptide C ₁₈ (4.6 × 250 mm) columns. As a result, the natural peptide and the synthetic peptide were eluted at the same time. Therefore, it can be said that the two peptides have the same chemical component.

Example 5 Determination of Relaxation Activity of Asteripectin on Starfish DRM

The relaxation activity of Asteripectin purified from starfish was measured using DRM of starfish in the same manner as in Example 2, and as a comparative substance, FMRFamide derived from S1 and S2 derived from epidermal animals and mollusks. And FLMFamide were used. Relaxation activity was expressed as% relative relaxation to maximal contraction of ^10-6 M acetylcholine. On the other hand, the magnitude of the response was compared as ED ₅₀ (concentration when representing 50% of the highest relaxation) and E _max (% relaxation at 10 ⁻⁵ M). ED ₅₀ values were calculated using the least square method, and statistical processing was done by student's t-test. All response values are expressed as means se se.

As a result, as shown in Figure 8, Asteripectin had a threshold value of 0.59% at 10 ^-10 M, and showed a maximum relaxation activity of 120.0 ± 7.0% at 10 ^-5 M. In addition, S1 showed a threshold value of 2.4% at 10 ⁻¹⁰ M, but showed a relaxation activity of 45.2 ± 2.4% at 10 ⁻⁵ M. In addition, S2 exhibited a threshold value of 2.6% at 10 ⁻⁷ M and a relaxation activity of about 15% at 10 ⁻⁵ M, which was lower than that of S 1. In addition, FLRFamide and FMRFamide showed little activity. The magnitude of the activity of these neuronal peptides was Asteripectin >>S1>S2>FLRFa> FMRFa.

Example 6 Identification of Active Sites of Peptides

In order to identify the smallest unit showing relaxation activity among the 16 amino acids constituting Asteripectin, peptides in which the N- or C-terminus of Asteripectin were cut were synthesized in the same manner as in Example 5.

The relaxation activity of the synthesized peptide was measured using the DRM of the starfish in the same manner as in Example 2 to confirm the shortest amino acid sequence showing the relaxation activity.

N-terminal and C-terminal Derivatives of Asteripectin (AP) Peptide Sequence N-terminal analogs AP (2-16)     G-K-G-G-A-Y-D-P-L-S-A-G-F-T-D (SEQ ID NO: 9) AP (3-16)       K-G-G-A-Y-D-P-L-S-A-G-F-T-D (SEQ ID NO: 10) AP (4-16)         G-G-A-Y-D-P-L-S-A-G-F-T-D (SEQ ID NO: 11) AP (5-16)           G-A-Y-D-P-L-S-A-G-F-T-D (SEQ ID NO: 12) AP (6-16)             A-Y-D-P-L-S-A-G-F-T-D (SEQ ID NO: 13) AP (7-16)               Y-D-P-L-S-A-G-F-T-D (SEQ ID NO: 14) C-terminal analogs AP (1-15)   F-G-K-G-G-A-Y-D-P-L-S-A-G-F-T (SEQ ID NO: 15) AP (1-14)   F-G-K-G-G-A-Y-D-P-L-S-A-G-F (SEQ ID NO: 16) AP (1-13)   F-G-K-G-G-A-Y-D-P-L-S-A-G (SEQ ID NO: 17) AP (1-12)   F-G-K-G-G-A-Y-D-P-L-S-A (SEQ ID NO: 18) AP (5-14)           G-A-Y-D-P-L-S-A-G-F (SEQ ID NO: 2)

As a result of measuring the relaxation activity of the synthesized peptide, AP (2-16), AP (3-16) and AP (4-16), which are N-terminal derivatives, AP (1-15) and AP, which are C-terminal derivatives, were measured. Since no significant change in relaxation activity was observed in (1-14) compared to Asteripectin, the fifth to fourteenth residues (AP (5-14)) were defined as the minimum residues showing activity (SEQ ID NO: 2).

Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

The present invention has the effect of providing a novel neuronal peptide having smooth muscle relaxation activity, and has the effect of providing a nucleic acid including DNA and RNA encoding the neuronal peptide.

The peptide of the present invention is expected to be useful for use as a muscle relaxant because it is composed of a small number of amino acids while showing a stronger relaxation activity than the previously known relaxation active peptides S1 and S2.

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Claims (10)

Neural peptides having smooth muscle relaxation activity having the amino acid sequence of SEQ ID NO: 2. The meptide according to claim 1, which has an amino acid sequence represented by SEQ ID NO: 1. The peptide of claim 1, wherein the C-terminus is carboxylated or amided. The peptide according to claim 1, wherein the N-terminal amine group is substituted with an alkyl or acetyl group. The peptide of claim 1, wherein the amino acid sequence is L-type and / or D-type. delete Recombinant vector comprising a DNA encoding a peptide of SEQ ID NO: 2. A transformed microorganism obtained by introducing the recombinant vector of claim 7 into a bacterium, yeast or fungus. A method for producing a neuronal peptide having smooth muscle relaxation activity, comprising culturing the transformed microorganism of claim 8. A pharmaceutical composition for improving any one or more symptoms selected from the group consisting of bronchial asthma, hypertension, diuresis and erectile dysfunction containing the peptide of any one of claims 1 to 5.

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