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Vasoactive intestinal polypeptide precursors have highly potent bronchodilatory activity
Peptides,Vol. 12, pp. 131-137. ©PergamonPressplc, 1991. Printedin the U.S.A.
0196-9781/91 $3.00 + .00
Vasoactive Intestinal Polypeptide Precursors Have Highly Potent Bronchodilatory Activity O S A M U ITO A N D SHINRO T A C H I B A N A
Eisai Tsukuba Research Laboratories, Eisai Company Ltd. 5-1-3, Tokodai, Tsukuba-shi, Ibaraki, 300-26 Japan Received 5 July 1990
ITO, O. AND S. TACHIBANA. Vasoactive intestinalpolypeptide precursors have highly potent bronchodilatory activity. PEPTIDES 12(1) 131-137, 1991.--We studied the structure-activityrelationships of vasoactive intestinal polypeptide (VIP) to determine whether there were active forms of C-terminal-free VIP, so that suitable structures could be identified and produced by recombinant technology. We found that some presumptive VIP precursors prepared by solid-phase synthesis exhibited a higher biological activity than natural VIP both in vitro and in vivo, although we could not determine the actual active fragment. VIP-GIyLys-OH and VIP-Gly-Lys-Arg-OH,which were extended from the C-terminal of mature VIP, demonstrated a respective 210% and 160% increase in bronchodilatory activity in comparison to the activity of natural VIP. Furthermore, these peptides exhibited a respective 110% and 130% increase in hypotensive activity when compared with VIP itself. VIP
Precursor
Bronchodilation
Hypotension
VASOACTIVE intestinal polypeptide (VIP) is an octacosapeptide originally isolated from the porcine duodenum by Said and Mutt in 1972 (30). Since the VIP sequence closely resembles those of secretin and glucagon, it was classified among the so called "secretin-glucagon family of peptides." It is known that VIP is widely distributed not only in the gastrointestinal tract where it was first identified, but also in the peripheral and central nervous systems, genital organs, and respiratory tract (8, 20, 24, 29). Furthermore, VIP demonstrates a high level of conservation of its amino acid sequence among many species (2-4, 9-11, 24, 25, 36) (as shown in Fig. 1). VIP has various biological effects on many organs and tissues, especially in the respiratory tract (23). There are only a few known peptides with a bronchodilatory activity and among a group including PHI, GRF, and hANP, VIP is the most potent bronchodilator (22,27). VIP immunoreactive nerves have been found in mammalian airways and pulmonary vessels (8) and VIP receptors recently have been found in human lung membranes (21). These data suggest that VIP might play an important physiological role in the pulmonary system, prompting investigation of its therapeutic potential in asthma. We considered that one of the bottlenecks to overcome before clinical application would be how to provide sufficient quantities of VIP at reasonable cost, since gene manipulation techniques cannot be directly utilized to produce C-terminal amidated peptides like VIP. Unfortunately, the active fragment of C-terminal free VIP has not yet been identified (1,37). On the other hand, the precursor of human VIP has been deduced from its DNA sequence to have a Gly-Lys-Arg sequence after the C-terminal amidated peptide (18,26). Mature VIP is presumed to be synthesized at f'n'st by processing at dibasic cleavage sites, followed by removal of the Arg and/or Lys residue and transformation of the exposed C-terminal Gly to the amide group (as shown in Fig. 2). The aim of this study was to fred C-terminal-free VIP ana-
logues with biological activity by investigating the structure-activity relationships of various peptides. Some of our results were reported at the Japan Symposium on Peptide Chemistry in 1987 (33). METHOD
Materials All peptides were prepared by the conventional solid-phase peptide synthesis method using a Beckman peptide synthesizer (Type 990). Resins coupled with Boc-amino acids and p-methylbenzhydrylamine resin were purchased from Peninsula Labs. (Belmont, CA). All amino acid derivatives and synthesis reagents were obtained from the Protein Research Foundation (Mino, Osaka), except for Boc-Met(O) which was from Kokusan Chemical Works (Tokyo). The Boc group was used for a-amino group protection, and the side chain protecting groups were as follows: His(Tos), Ser(Bzl), Thr(Bzl), Asp(OcHex), Tyr(BrZ), Lys(C1Z), and Arg(Tos). All other reagents used were of reagent grade. Commercially obtained Boc-amino acid derivatives, the resins with bound Boc-amino acids, p-methylbenzhydrylamine (PMBHA) resin, the solvents, and the reagents for synthesis were used without further purification. Natural VIP was extracted from porcine duodenums at our laboratory (32) and further purified by high performance liquid chromatography (HPLC). Natural VIP was used not only as the standard in bioassays, but also for the structural confirmation of the synthesized peptides.
Synthesis of Peptides Synthesis scale, with few exceptions, was achieved in 0.5 mmoles and couplings were carried out by the dicyclohexylcarbodiimide/1-hydroxybenzotriazole(DCC/HoBt) method. A five-fold
131
132
ITO AND TACHIBANA
human, porcine, bovine, rat, canine II-IISDAVFTDNYTRLRKQMAVKKYLNS 1
5
10
15
I L N-HH2 20
25
---C-terminal amino a c i d - - G 1 y - -
28
L y s --A (Arg)
r g---
(Lys) Enzymatic Cleavage
FIG. 1. Amino acid sequence of V1P.
Enzymatic Cleavage excess of blocked amino acids and reagents was utilized according to the protocol shown in Fig. 3. Arginine was coupled by the standard DCC method to prevent lactam formation of the N imgroup. His was also coupled by the standard DCC method to prevent destruction of Tos. Coupling was monitored by the standard ninhydrin test (19) and coupling reactions were repeated in an attempt to produce improved coupling when the ninhydrin test proved to be positive. Peptides were acetylated by acetic anhydride if the ninhydrin test failed to demonstrate an improvement after repeating of the coupling reaction. At the completion of synthesis, the N-ct-Boc group was removed with 40% trifluoroacetic acid (TFA) in dichloromethane according to steps 1-9 of the protocol, and the protected peptide resin was dried in vacuo for 18 hours at room temperature. After construction of the desired peptide chain, cleavage of the peptide from the resin and concomitant deprotection were achieved by a single treatment with anhydrous hydrogen fluoride (HF). Protected peptide resin was stirred with HF (20 ml) containing 10% anisole and 5% dimethyl sulfide at - 20°C for one hour. After removal of HF, the residue was washed with diethyl ether by decantation. The crude precipitate was transferred to a glass filter with diethyl ether, washed once more with an excess of diethyl ether, and then dried in vacuo. The mixture of crude products was extracted with 10% acetic acid at room temperature for several minutes, filtered through a fritted funnel, and lyophilized. (A typical chromatogram of crude product is shown in Fig. 4.) At the first purification, the crude peptide was dissolved in 0.05 M ammonium acetate buffer (pH 7.0), loaded into a carboxymethyl-cellulose (CM-C) column (Whatman CM-52, 2.5 x 30 cm), and eluted with a linear 0.05 M to 0.5 M ammonium acetate gradient (pH 7.0). The main fractions were then combined and lyophilized. The second purification was achieved by semipreparative reverse-phase HPLC (RP-HPLC), using a C18 (octadecyl silica; ODS) column (YMC ODS 5 l~m A-343, 20 × 250 mm, Kyoto) with a linear gradient of 20% to 40% acetonitrile (CH3CN) containing 0.01 N hydrochloric acid (HC1) or 1% TFA. Final purification to homogeneity was achieved by elution through a semipreparative HPLC C18 column (YMC ODS A-323 5 i.tm 10 × 250 mm) with a linear gradient of 1% to 45% acetonitrile containing 0.01 N acetic acid (AcOH). The purified peptide was then lyophilized. Homogeneity of the final purified peptides was assessed by analytical RP-HPLC (YMC ODS 5 txm AM-303, 4 . 6 x 250 mm and Nucleosil C18 5 Ixm 4 . 6 × 2 5 0 mm) with a TFA/CH3CN solvent system. The amino acid composition was calculated using a Hitachi amino acid analyzer Model 835 equipped with a system amino acid computing integrator, after the purified product was hydrolyzed with 6 N HC1 containing 1% phenol for 24 hours at 110°C in vacuum tubes. The structure of each synthesized peptide was confirmed by fast atom bombardment (FAB) mass spectrometry performed using a double focusing nmss spectrometer (JOEL, type JMS-HX100, Tokyo). For structural analysis, peptide mapping was performed by digestion with TPCK-treated trypsin (Sigma). Natural VIP and synthetic peptides (10 nmoles) in 50 mM phosphate buffer (100 Ixl, pH 7.4) were incubated together with TPCK-treated trypsin (5 Ixl of a 0.1 mg/ml solution) for 2 hours at 37°C, and then boiled for 5
---C-terminal amino acid- - G I y - - O H Enzymatic Conversion
---C-terminal amino a c i d - - N H 2 FIG. 2. C-terminal amidated peptide.
min to stop the digestion. Thereafter, the solution was directly loaded onto an analytical RP-HPLC column (ODS) to compare it with natural VIP and to confirm the modifications. Typical chromatograms of the fragments are shown in Fig. 6, and each fragment was identified by amino acid analysis.
Biological Assays Bronchodilatory activity. Bronchodilatory activity was determined by the relaxation of isolated guinea pig tracheal smooth muscle. A male guinea pig weighing about 300 g was bled to death from the jugular vein and then thoracotomized. Immediately after the thoracotomy, 3 cm of the trachea was removed and immediately transferred to Krebs solution (NaCI: 118.1 raM, KCI: 4.69 mM, MgSO4:0.60 mM, KH2PO4:0.92 mM, NaHCO3:25 raM, CaC12:2.52 mM, and glucose: 0.2%). This tracheal segment was cut into seven equal-sized rings and these cartilage rings were connected to one another adjacent to the tracheal smooth muscle with cotton yam. Subsequently, the cartilage opposite the smooth muscle was cut, so that the smooth muscle portions could be connected to each other. This preparation was then suspended in an isothermic tissue bath (7 ml). The lower part of the preparation was fixed, while the upper part was connected to an isotonic transducer with a load of 0.5 g, which was used to determine the extent of the relaxation reaction. Krebs solution containing 6.5 x 10 6 M histamine was used to precontract the smooth muscle. This solution was aerated with a mixture of 95% oxygen and 5% carbon dioxide and was maintained at 37°C. The tissues were superfused with the solution at a flow rate of 3.3 ml/min. The relaxation reaction was shown to be proportional to the amount of the test agent that was added. Utilizing this relationship, the relaxation of the tracheal smooth muscle by various synthetic peptides was compared with that achieved by VIP. The VIP standard (10 -5 M) was added in 20 it1 and 40 ILl (200 pmoles and 400 pmoles) or 10 ixl and 20 Ixl aliquots. The peptides under study were also used at 10 -5 M, with the exception that N-terminaldeleted fragments were assayed at 10 - 3 M, and were added as aliquots of 10 Ixl and 20 p.1. The area (height × time) of the relaxation curve for each peptide was determined, and specific activities were obtained by taking the area for VIP as 100% and calculating the relative ratio for the area of each peptide. The relaxation reactions were thus compared by the four-point assay method using the above-mentioned procedure (5,28). Hypotensive effect. The hypotensive effect of each peptide was determined by the changes in carotid artery pressure in a rat. A Sprague-Dawley male rat weighing 200 g was anesthetized by the intraperitoneal administration of 48% urethane at a dose of 2.7
VIP PRECURSORS AND BRONCHODILATION
133
Reagent
Step
Time
CH2C12 wash 3 times 40% TFA/CH2C12 deprotection 40% TFA/CH2C12 deprotection CH2CI2 wash 5 times EtOH wash 3 times CH2C12 wash 5 times 10% TEA/CH2C12 neutralization 10% TEAJCH2CI2 neutralization CH2CI2 wash 5 times Boc-amino acid/CH2C12 delivery 5-fold excess 0.5 M DCC/CH2CI2 delivery 5-fold excess mix for coupling or until monitoring shows complete reaction CH2C12 wash 5 times Recoupling, if necessary, by repeating steps 6-13 CH2CI2 wash 5 times
1:
2: 3: 4: 5: 6: 7: 8: 9: 10: 11: 12: 13: 14: 15:
1 mln 1 man
20 nun 1 nun 1 nun 1 nun 1 nun 10 nun 1 nun
up to 120 min 1 min 1 min
FIG. 3. Protocol for solid-phase peptide synthesis.
ml/kg. The carotid artery was cannulated for the injection of each peptide and a transducer cannula for determining the blood pressure was also inserted into the carotid artery. Peptide samples (12.5 to 400 pmol/kg) were then administered into the carotid artery, and the changes in blood pressure were plotted with a polygraph to obtain dose-response curves. The amount of each sample required to produce a drop of 20 mmHg in blood pressure was determined from the curves. The amount for native VIP was regarded as 100%, and the specific activity of each sample was obtained by calculating the relative ratio.
RESULTS
Peptide Synthesis The crude peptides obtained by cleavage of the protected peptide resins each had a major peak on RP-HPLC analysis (Fig. 4). Synthetic peptides gave a single peak in analytical HPLC after final purification and the comparison of synthetic and natural VIP by analytical RP-HPLC showed that they had the same retention (RT) and eluted together on cochromatography (Fig. 5). The amino acid compositions of these peptides after hydrolysis were consistent with the theoretical values (Table 1). FAB mass spectrometry data also agreed with the theoretical predictions (data not shown). For sequence confirmation peptide mapping, peptides treated with TPCK-trypsin were compared with natural VIP using RP-HPLC, as shown in Fig. 6. The digestion of VIP and synthetic peptides by TPCK-trypsin yielded two C-terminal fragments, as expected from the location of the two lysines (positions
Statistical Methods The parallel line assay was used to estimate the relative potency and 95% confidence limits with the aid of a computer for bronchodilatory activity (13). Comparison of the hypotensive effect was made by A N O V A and Scheffe's multiple comparison.
TABLE 1 AMINO ACID COMPOSITIONS OF VIP AND ITS ANALOGUES AFTER HYDROLYSIS BY 6 N HC1 CONTAINING 1% PHENOL AT 110°C FOR 24 HOURS IN A VACUUM TUBE
Asp
Thr
Ser
Glu
Gly
Ala
Val
Met
Ile
Leu
Tyr
Phe
Lys
His
Arg
Natural VIP Synthetic VIP (theo)
5.0 5.0 (5)
1.9 2.0 (2)
1.8 1.8 (2)
1.1 1.1 (1)
0.2 0 (0)
2.0 2.1 (2)
1.9 2.0 (2)
0.9 1.0 (1)
1.0 1.0 (1)
3.1 3.0 (3)
1.9 2.0 (2)
1.0 1.0 (1)
3.2 3.1 (3)
0.9 1.1 (1)
2.1 2.0 (2)
VIP-OH 1-27-OH 2-28-NH 2 3-28-NH z [Met~7(O)]VIP [Leu17]VIP VIP-G-OH VIP-G-K-OH VIP-G-K-R-OH
5.0 4.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0
1.9 1.9 2.0 1.9 1.9 2.0 1.9 1.9 1.8
1.8 1.7 1.8 0.9 1.7 1.8 1.8 1.8 1.7
1.1 1.1 1.1 1.1 1.1 1.1 1.1 1.1 1.0
0 0 0 0 0 0 1.l 1.1 1.0
2.1 2.0 2.1 2.1 2.0 2.3 2.1 2.1 2.0
2.0 2.0 2.0 2.0 2.0 2.1 2.0 2.0 1.9
0.8 1.0 0.8 1.0 0.6 0 1.0 1.0 1.0
1.0 1.0 1.0 1.0 1.0 0.9 1.1 1.0 0.9
3.1 3.2 3.1 3.1 3.2 4.1 3.1 3.1 3.1
2.0 2.0 2.0 2.0 2.0 2.1 2.0 2.0 2.0
1.0 1.0 1.0 1.0 1.0 1.1 1.0 1.0 1.0
3.0 3.1 3.0 3.0 3.2 3.2 3.0 4.0 4.0
1.1 1.1 0 0 1.1 0.9 1.1 1.1 1.1
2.0 2.0 2.0 2.0 2.0 2.2 2.0 2.0 3.1
One nmole of each peptide was hydrolyzed.
134
ITO AND TACHIBANA
~40
,,~ 0.2<.%
0.4-
A
E C)
-30
cq
~
,~ o,1-
0.3-
I
~(..) z
+
E
g
*zl
F~
-20
4O
3 o.2-
I
10
'~
0.2
z
20
c
u')
~o
3'0
,~o
Time (mln)
8
== ,,,Q
o
FIG. 4. Reverse-phase HPLC analysis of crude VIP-G-K-OH on a C18 column. A linear gradient from 22.5% to 37.5% acetonitrile containing 0.1% TFA was used for 40 min at a flow rate of 1.0 ml/min.
,,D ,,¢
-30
I
,~ 0.1-
1~,
3'0 -40
30
0.1!
20 T i m e (rain)
O
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C) I ~0 z
-20
. L~"
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I
Time (min)
20
30 -40
,,m0.2" 20 and 21 in the peptide sequence). Synthetic VIP fragments were revealed to have exactly the same chromatogram as natural VIP by RP-HPLC analysis. C-terminal fragments originating from Cterminal-modified peptides revealed different RTs to the natural fragment, while other fragments were consistent with the same portions of VIP. These tryptic fragments were subjected to amino acid analysis after isolation of each peak by RP-HPLC and hydrolysis with 6 N HC1. Their amino acid compositions were consistent with the expected compositions of the tryptic fragments derived from the peptides (Table 2).
Biological Studies
I
-30
z
o
20
,.Q ,,¢
i 10
20 Time
30
(rain)
ii :)
-40
~0.2-
The potency of the synthetic peptides was evaluated by assessing guinea pig tracheal smooth muscle relaxation in comparison with VIP in vitro and the hypotensive effect in the rat in vivo. Table 3 shows the tracheal relaxation achieved by the synthetic peptides. Among the fragments that were shorter than mature VIP, VIP-OH (which was C-terminal free instead of amidated) had 30% of activity of VIP, and VIP(1-27)-OH (which had deletion of the C-terminal Asn residue) had only 4.5% of the activity of VIP. N-terminal-deleted peptides (2-28 and 3-28) showed under 1% relative potency compared with natural VIP. Among the C-terminal-extended peptides, VIP-Gly-OH showed only 50% of the bronchodilatory activity of VIP, but VIP-Gly-Lys-OH and VIP-Gly-Lys-Arg-OH had, respectively, 210% and 160% of the activity of native VIP. The relative potency of [MetlT(O)]VIP was decreased to one-third of that of the original VIP and [Leu~7]VIP had 70% of the original bronchodilatory activity. Table 4 shows the hypotensive effect on rat blood pressure in vivo. The ED2o mmHg, which means the dose required to reduce the blood pressure of an anesthetized rat by 20 mmHg, was 81 --- 10 pmol/kg for the VIP standard. Relative potency was calculated using this ED2o mmHg value as 100%. The ED2o mmHg for VIP-OH, VIP(1-27)-OH, VIP(2-28)-NH2, and VIP(3-28)-NH 2 was 105---8, 5 1 5 - - 2 6 9 , 937---160, and > 1 0 0 0 pmol/kg (mean--. SEM), respectively. In the case of C-terminal-extended peptides, VIP-Gly-OH, VIP-Gly-Lys-OH, and VIP-Gly-Lys-Arg-
0 "l-
/
,~ o.1
/
u')
t'3
~
30 0.1-
Z
- 20 I 10
I 20 Time
I 30
(rain)
FIG. 5. Reverse-phase HPLC analysis of synthetic and natural VIP on a C18 column. A lineargradientfrom 22.5% to 37.5% acetonitrilecontaining 0.1% TFA was used for 40 min at a flow rate of 1 ml/min. (A) Natural VIP (1 nmole). (B) SyntheticVIP (1 nmole). (C) Cochromatography of natural and synthetic VIP (equal quantities). (D) VIP-G-K-OH (1 nmole). OH had 110--.22, 71---8, and 63---3 pmol/kg. Substitution at 17 position {[MetlT(O)]VIP and [Leu 17 ]VIP} gave potencies of 131-4-47 and 1 0 3 - 14 pmol/kg, respectively. DISCUSSION
The results presented in this study show that the peptides pre-
VIP PRECURSORS AND BRONCHODILATION
135
TABLE 2 AMINO ACID COMPOSITIONS OF VIP-G-K-OHAND TRYPSIN FRAGMENTS AFIT_,RHYDROLYSIS BY 6 N HCl CONTAINING 1% PHENOL AT ll0*C FOR 24 HOURS IN A VACUUMTUBE
VIP-G-K-OH (theo) Fr.1 Fr.2 Fr.3 Fr.4 Fr.5 Fr.6 Fr.7 Fr.8 Fr.9 Fr.10
Asp
Thr
Ser
Glu
Gly
Ala
Val
Met
lie
Leu
Tyr
Phe
Lys
His
Arg
5.0 (5)
1.9 (2)
1.8 (2)
1.1 (1)
1.1 (1)
2.1 (2)
2.0 (2)
1.0 (1)
1.0 (1)
3.1 (3)
2.0 (2)
1.0 (1)
4.0 (4)
1.1 (1)
2.0 (2)
1.0 1.0 1.0 1.0 1.0 1.0 1.0 3.0 2.0 2.0
1.9
0.9 0.8 0.8
0.94 1.08 0.98 0.98 0.90 1.04
1.06 1.23 1.02 1.06 1.05 1.0
0.76 0.85 0.94 0.94 0.90
1.01 0.99
0.99 0.97
1.02 1.03
0.81 2.65 1.69 1.81 1.00 1.29 0.98 1.01 1.00
2.05 2.03
1.00
0.87
1.02
1.83 0.93
One nmole of each peptide was hydrolyzed.
pared by the solid-phase method had the desired sequences and purity. Although the biological activity of a few VIP fragments has been evaluated previously (1, 15, 37), it remained unknown that peptides with the C-terminal free and the almost same size as VIP needed to have a satisfactory biological activity (38). As our purpose was to identify bioactive C-terminal-free VIP, we first tried to shorten the peptide. Therefore, we synthesized the C-terminal amide hydrolyzed peptide VIP-OH and the C-terminal-deleted peptide VIP(1-27)-OH, and compared them with N-terminaldeleted peptides [VIP(2-28) and VIP(3-28)] with respect to their bronchodilatory and hypotensive activities. VIP-OH had almost the same hypotensive activity as the original VIP, whereas its bronchodilatory activity was decreased to one-third of that of VIP. VIP(1-27)-OH maintained one-fifth of the hypotensive activity of the original VIP, whereas its bronchodilatory potency was only
4.5% of that of VIP. As shown in Table 3, bronchodilatory activity was lost from the N-terminal-deleted peptides when the Nterminal His was deleted. On the other hand, their hypotensive activity gradually decreased in proportion to the loss of chain length. In early studies, Bodanszky et al. claimed that some VIP fragments possessed considerable biological activity (1) and Fournier et al. reported fragments with hypotensive activity (15). Our results in regard to hypotensive activity support their find-
ot
50
i
25
0.1
0
o[
TABLE 3 RELATIVE POTENCY OF GUINEA PIG TRACHEAL RELAXANT ACTIVITY IN VITRO
B
I
50
Bronchodilatory Activity Synthetic VIP A) VIP-OH VIP(1-27)-OH VIP(2-28)-NH2 VIP(3-28)-NH2
25
Defined as 100%
o
30% (5-35%)* 4.5% (0.4--5.5%)* < 1.0% < 1.0%
n= 8 n= 8 n= 8 n= 8
VIP-G-OH VIP-G-K-OH VIP-G-K-R-OH
50% (40-60%)* 210% (190-240%)* 160% (140--190%)*
n = 16 n = 16 n= 8
C) [MetI7(O)]VIP [Leu~7]VIP
30% (20-40%)* 70% (65-85%)*
n= 8 n= 8
B)
"~
0.1 .
.
.
.
.
.
.
.
.
.
" "
25
o.
A) Comparison of fragments with C- and N-terminal deletions. B) Comparison of precursors. C) Comparison of substitution at position 17. *95% confidence limits.
~o
o
0
10
20 Tlme (rnln)
30
FIG. 6. Reverse-phase HPLC analysis of trypsin-digested fragments of VIP and its analogues on a C18 column. Each peptide (10 nmoles) was digested with 20 p,g of trypsin (TPCK-treated) in 100 p,1 of 0.1 M phosphate buffer (pH 7.4) at 37°C for 6 h. Half of the reaction mixture was loaded directly into the column, and separated using a linear gradient of 1% to 48% acetonitrile containing 0.1% TFA for 40 rain at flow rate of 1.0 ml/min. (A) natural VIP. (B) synthetic VIP. (C) VIP-Gly-Lys-OH.
136
ITO AND TACHIBANA
TABLE 4 COMPARISON OF HYPOTENSIVE ACTIVITY IN VIVO
Synthetic VIP A) VIP-OH VIP(1-27)-OH VIP(2-28)-NH 2 VIP(3-28)-NH 2
ED2o mmHg* (pmol/kg) (n_-----8)
Relative PotencyS-
81 ~ 10
100%
105 515 937 >
+ 8 -+ 269§ --- 160§ 10000
80% 20% 10% < 1%
B) VIP-G-OH VIP-G-K-OH VIP-G-K-R-OH
110 - 22 71 --- 8:~ 63 _+ 3:~
70% 110% 130%
C) [MetlT(o)]vIP [LeuaT]VIP
131 --- 47 103 --- 14
60% 80%
A) Effect of fragments with C- and N-terminal deletions. B) Effect of precursors. C) Effect of substitutions at position 17. *ED2o mmHg means the dose of the sample required to produce a drop of 20 mmHg in the blood pressure of an anesthetized rat. Values are the means ---SE. tVIP as 100%. ~p<0.05, §p<0.01.
ings, but our data on bronchodilatory activity are inconsistent with previous observations. Our results suggest that the essential structures for producing bronchodilation were more strictly limited than those related to the hypotensive effect of VIP. Although these findings confirmed that there should be no smaller C-terminalfree peptides than VIP itself, we considered that there may be active forms of C-terminal-free VIP because VIP-OH had a comparable activity to natural VIP. Accordingly, we synthesized several C-terminal-extended precursors of VIP: VIP-Gly-OH, VIPGly-Lys-OH, and VIP-Gly-Lys-Arg-OH. The latter two synthetic
peptides showed an unexpectedly high bronchodilatory activity (Table 3). On the other hand, the precursor VIP-GIy-OH, which represents VIP just before maturation, had only about half the potency of VIP in bronchorelaxant assays, while displaying a hypotensive activity equal to 80% of that of mature VIP. These resuits raised an interesting question as to whether bioactive Cterminal-extended precursors of VIP play a role in mammalian physiology. It is quite unusual for precursors to possess higher bioactivities than the mature form of a peptide. Recently, C-terminal-extended secretin precursors (secretin-Gly-OH and secretin-Gly-Lys-Arg-OH) were isolated from porcine upper intestinal tissue, and the latter peptide exhibited a substantially higher bioactivity than secretin itself (16). In the case of VIP, other reports also support our findings and it may be that variations in the structure of VIP led to its tissue selectivity (12, 13, 17, 31, 38). As secretin and VIP belong to the same family of gastrointestinal hormones, there may be a similar reason for the existence of highly active precursors that is at present unknown. It has been reported that the Met 17 residue of VIP is easily oxidized to produce [MetIT(o)]vIP (37). However, there has been no quantitative and comparative data available on the bioactivities of these peptides. We synthesized the oxidized form Met17(O) by the solid-phase method, using Boc-Met(O) instead of Boc-Met. The presence of methionine sulfoxide in the peptide sequence was confirmed by HPLC, which distinguished the oxidized form from VIP (results not shown). The bronchodilatory activity of the oxidized peptide was decreased to one-third of that of natural VIP, but the hypotensive potency only decreased slightly. This finding was in good accord with the previously mentioned suggestion that bronchodilation seems to demand a strict structure while the structure producing the hypotensive effect is permissive. [Leu~7]VIP had a potency nearly equivalent to VIP itself for both bronchodilatory and hypotensive activity. PHI and PHM, which are closely related in sequence and biological properties to VIP, have Leu in position 17 of their sequences instead of the Met ~7 of VIP and are encoded by the same DNA (18, 32, 33). Furthermore, it was reported recently that helodermin, which is a C-terminal extended VIP-like peptide and has Leu 17 in its sequence, also had bronchodilatory properties (13). These and our findings suggest that C-terminal-free VIP with Leu 17 may have a high level of both bronchodilatory and hypotensive activity.
REFERENCES 1. Bodanszky, M.; Henes, J. B.; Yiotakis, A. E.; Said, S. I. Synthesis and pharmacological properties of the N-terminal decapeptide of the vasoactive intestinal peptide (VIP). J. Med. Chem. 20(11):14611464; 1977. 2. Bodanszky, M.; Bodanszky, A.; Said, S. I. Synthesis of the avian vasoactive intestinal peptide (VIP). Fed. Proc. 37:829; 1978. 3. Carlquist, M.; Mutt, V.; Jornvall, H. Isolation and characterization of bovine vasoactive intestinal peptide (VIP). FEBS Lett. 108(2): 457-460; 1979. 4. Carlquist, M.; McDonald, T. J.; Go, V. L. W.; Bataille, D.; Johansson, C.; Mutt, V. Isolation and amino acid composition of human vasoactive intestinal polypeptide (VIP). Horm. Metab. Res. 14:2829; 1982. 5. Castillo, D. B. The tracheal chain 1. A preparation for the study of antispasmodics with particular reference to bronchodilator drugs. J. Pharrnacol. Exp. Ther. 90:104-109; 1947. 6. Colombo, R. A new solid-phase synthesis of porcine vasoactive intestinal peptide using Na-9-fluorenylmethyloxycarbonyl amino acids. Experientia 38:773-775; 1982. 7. Coy, D. H.; Gardner, J. Solid-phase synthesis of porcine vasoactive intestinal peptide. Int. J. Pept. Protein Res. 15:73-78; 1980. 8. Dey, R. D.; Shannon, W. A., Jr.; Said, S. I. Localization of VIPimmunoreactive nerves in airways and pulmonary vessels of dogs,
cats, and human subjects. Cell Tissue Res. 220:231-238; 1981. 9. Dimaline, R.; Reeve, J. R., Jr.; Shivelly, J. E.; Hawke, D. Isolation and characterization of rat vasoactive intestinal peptide. Peptides 5: 183-187; 1984. 10. Du, B. H.; Eng, J.; Hulmes, J. D.; Chang, M.; Pan, Y. C. E.; Yalow, R. S. Guinea pig has unique mammalian VIP. Biochem. Biophys. Res. Commun. 128(3):1093-1098; 1985. 11. Eng, J.; Du, B.-H.; Raufman, J.-P.; Yalow, R. S. Purification and amino acid sequences of dog, goat and guinea pig VIPs. Peptides 7(Suppl. 1):17-20; 1986. 12. Fahrenkrug, J.; Ottesen, B.; Palle, C. Non-amidated forms of VIP (glycine-extended VIP and VIP-free acid) have full bioactivity on smooth muscle. Regul. Pept. 26:235-239; 1989. 13. Finney, D. J. Statistical method in biological assay (2nd edition). New York: Hafner Publishing Co.; 1964. 14. Foda, H. D.; Said, S. I. Helodermin, a C-terminal extended VIP-like peptide, evokes long-lasting tracheal relaxation. Biomed. Res. 10(1): 107-110; 1989. 15. Fournier, A.; Saunders, J. K.; St-Pierre, S. Synthesis, conformational studies and biological activities of VIP and related fragments. Peptides 5:169-177; 1984. 16. Gafvelin, G.; Carlquist, M.; Mutt, V. A proform of secretin with high secretin-like bioactivity. FEBS Lett. 184(2):347-352; 1985.
VIP PRECURSORS AND BRONCHODILATION
17. Gafvelin, G.; Andersson, M.; Dimaline, R.; Jornvall, H.; Mutt, V. Isolation and characterization of a variant form of vasoactive intestinal polypeptide. Pcptides 9:469--474; 1988. 18. Itoh, N.; Obata, K.; Yanaihara, N.; Okamoto, H. Human preprovasoactive intestinal polypeptide contains a novel PHI-27-1ike peptide, PHM-27. Nature 304(11):547-549; 1983. 19. Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Color test for detection of free terminal amino groups in the solid-phase synthesis of peptide. Anal. Biochem. 34:595-598; 1970. 20. Larsson, L.-I.; Fahrenkrug, J.; Schafflitzky de Muckadell, O.; Sundler, F.; Hakaqnson, R.; Rehfeld, J. Localization of vasoactive intestinal polypeptide (VIP) to central and peripheral neurons. Proc. Natl. Acad. Sci. USA 73:3197-3200; 1976. 21. Leys, K.; Morice, A. H.; Madonna, O.; Sever, P. S. Autoradiographic localisation of VIP receptor in human lung. FEBS Lett. 199(2): 198-202; 1986. 22. Lundberg, J. M.; Fahrenkrug, J.; Hokfelt, T.; Martling, C. R.; Larsson, O.; Tatemoto, K.; Anggard, A. Co-existence of peptide HI (PHI) and VIP in nerves regulating blood flow and bronchial smooth muscle tone in various mammals including man. Peptides 5:593-606; 1984. 23. Matsuzaki, Y.; Hamasaki, Y.; Said, S. I. Vasoactive intestinal peptide: A possible transmitter of non-adrenergic relaxation of guinea pig airways. Science 210:1252-1253; 1980. 24. Mutt, V.; Said, S. I. Structure of the porcine vasoactive intestinal octacosapeptide. Eur, J. Biochem. 42:581-589; 1974. 25. Nilsson, A. Isolation, amino acid composition and terminal amino acid residues of the vasoactive octacosapeptide from chicken intestine. Partial purification of chicken secretin. FEBS Lett. 47:284-289; 1974. 26. Nishizawa, N.; Hayakawa, Y.; Yanaihara, N.; Okamoto, H. Nucleotide sequence divergence and functional constraint in VIP precursor mRNA evolution between human and rat. FEBS Lett. 183:55-59; 1985. 27. O'Donnell, M.; Garippa, R.; Welto, A. F. Relaxant activity of atriopeptins in isolated guinea pig airway and vascular smooth muscle. Peptides 6:597-601; 1985. 28. Perry, W. L. M. Pharmacological experiments on isolated prepara-
137
tions. London: Livingstone Ltd.; 1968. 29. Polak, J. M.; Bloom, S. R. Localization and measurement of VIP in the genitourinary system of man and animals. Peptides 5:225-230; 1984. 30. Said, S. I.; Mutt, V. Isolation from porcine intestinal wall of vasoactive octacosapeptide related to secretin and glucagon. Eur. J. Biochem. 28:199-204; 1972. 31. Simoncsits, A.; Tjomhammar, M. L.; Kalman, M.; Cserpan, I.; Gafvelin, G.; Bartfai, T. Synthesis, cloning and expression in Escherichia coli of artificial genes coding for biologically active elongated precursors of the vasoactive intestinal polypeptide. Eur. J. Biochem. 178:343-350; 1988. 32. Suzuki, S.; Araki, K.; Tachibana, S. Isolation of porcine vasoactive intestinal polypeptide and comparison of its biological activities with secretin and glucagon. Yakugaku Zasshi 99:172-179; 1979. 33. Tachibana, S.; Ito, O. Novel bioactive precursor analogues of vasoactive intestinal polypeptide (VIP)--Structure-activity relationships. In: Shiba, T.; Sakakibara, S., eds. Peptide Chemistry 1987. Osaka: Protein Research Foundation; 1988:481--486. 34. Tatemoto, K.; Mutt, V. Isolation and characterization of the intestinal peptide porcine PHI (PHI-27), a new member of the glucagonsecretin family. Proc. Natl. Acad. Sci. USA 78(11):6603--6607; 1981. 35. Tatemoto, K.; Jonvall, H.; McDonald, T. J.; Carlquist, M.; Go, V. L. W.; Johansson, C.; Mutt, V. Isolation and primary structure of human PHI (peptide HI). FEBS Lett. 174(2):258-261; 1984. 36. Wang, S. C.; Du, B. H.; Eng, J.; Chang, M.; Hulmes, J. D.; Pan, Y.-C. E.; Yalow, R. S. Purification of dog VIP from a single animal. Life Sci. 37:979-983; 1985. 37. Yanaihara, N.; Nokihara, K.; Yamamoto, K.; Hashimoto, T.; Yanaihara, T.; Ito, Z.; Yamaguchi, K.; Abe, K. Immunological aspects of secretin, substance P and VIP. Gastroenterology 72:803810; 1977. 38. Yiangou, Y.; Marzo, V. D.; Spokes, R. A.; Panico, M.; Morris, H. R.; Bloom, S. R. Isolation, characterization, and pharmacological actions of peptide histidine valine 42, a novel prepro-vasoactive intestinal peptide-derived peptide. J. Biol. Chem. 262:14010-14013; 1988.
0196-9781/91 $3.00 + .00
Vasoactive Intestinal Polypeptide Precursors Have Highly Potent Bronchodilatory Activity O S A M U ITO A N D SHINRO T A C H I B A N A
Eisai Tsukuba Research Laboratories, Eisai Company Ltd. 5-1-3, Tokodai, Tsukuba-shi, Ibaraki, 300-26 Japan Received 5 July 1990
ITO, O. AND S. TACHIBANA. Vasoactive intestinalpolypeptide precursors have highly potent bronchodilatory activity. PEPTIDES 12(1) 131-137, 1991.--We studied the structure-activityrelationships of vasoactive intestinal polypeptide (VIP) to determine whether there were active forms of C-terminal-free VIP, so that suitable structures could be identified and produced by recombinant technology. We found that some presumptive VIP precursors prepared by solid-phase synthesis exhibited a higher biological activity than natural VIP both in vitro and in vivo, although we could not determine the actual active fragment. VIP-GIyLys-OH and VIP-Gly-Lys-Arg-OH,which were extended from the C-terminal of mature VIP, demonstrated a respective 210% and 160% increase in bronchodilatory activity in comparison to the activity of natural VIP. Furthermore, these peptides exhibited a respective 110% and 130% increase in hypotensive activity when compared with VIP itself. VIP
Precursor
Bronchodilation
Hypotension
VASOACTIVE intestinal polypeptide (VIP) is an octacosapeptide originally isolated from the porcine duodenum by Said and Mutt in 1972 (30). Since the VIP sequence closely resembles those of secretin and glucagon, it was classified among the so called "secretin-glucagon family of peptides." It is known that VIP is widely distributed not only in the gastrointestinal tract where it was first identified, but also in the peripheral and central nervous systems, genital organs, and respiratory tract (8, 20, 24, 29). Furthermore, VIP demonstrates a high level of conservation of its amino acid sequence among many species (2-4, 9-11, 24, 25, 36) (as shown in Fig. 1). VIP has various biological effects on many organs and tissues, especially in the respiratory tract (23). There are only a few known peptides with a bronchodilatory activity and among a group including PHI, GRF, and hANP, VIP is the most potent bronchodilator (22,27). VIP immunoreactive nerves have been found in mammalian airways and pulmonary vessels (8) and VIP receptors recently have been found in human lung membranes (21). These data suggest that VIP might play an important physiological role in the pulmonary system, prompting investigation of its therapeutic potential in asthma. We considered that one of the bottlenecks to overcome before clinical application would be how to provide sufficient quantities of VIP at reasonable cost, since gene manipulation techniques cannot be directly utilized to produce C-terminal amidated peptides like VIP. Unfortunately, the active fragment of C-terminal free VIP has not yet been identified (1,37). On the other hand, the precursor of human VIP has been deduced from its DNA sequence to have a Gly-Lys-Arg sequence after the C-terminal amidated peptide (18,26). Mature VIP is presumed to be synthesized at f'n'st by processing at dibasic cleavage sites, followed by removal of the Arg and/or Lys residue and transformation of the exposed C-terminal Gly to the amide group (as shown in Fig. 2). The aim of this study was to fred C-terminal-free VIP ana-
logues with biological activity by investigating the structure-activity relationships of various peptides. Some of our results were reported at the Japan Symposium on Peptide Chemistry in 1987 (33). METHOD
Materials All peptides were prepared by the conventional solid-phase peptide synthesis method using a Beckman peptide synthesizer (Type 990). Resins coupled with Boc-amino acids and p-methylbenzhydrylamine resin were purchased from Peninsula Labs. (Belmont, CA). All amino acid derivatives and synthesis reagents were obtained from the Protein Research Foundation (Mino, Osaka), except for Boc-Met(O) which was from Kokusan Chemical Works (Tokyo). The Boc group was used for a-amino group protection, and the side chain protecting groups were as follows: His(Tos), Ser(Bzl), Thr(Bzl), Asp(OcHex), Tyr(BrZ), Lys(C1Z), and Arg(Tos). All other reagents used were of reagent grade. Commercially obtained Boc-amino acid derivatives, the resins with bound Boc-amino acids, p-methylbenzhydrylamine (PMBHA) resin, the solvents, and the reagents for synthesis were used without further purification. Natural VIP was extracted from porcine duodenums at our laboratory (32) and further purified by high performance liquid chromatography (HPLC). Natural VIP was used not only as the standard in bioassays, but also for the structural confirmation of the synthesized peptides.
Synthesis of Peptides Synthesis scale, with few exceptions, was achieved in 0.5 mmoles and couplings were carried out by the dicyclohexylcarbodiimide/1-hydroxybenzotriazole(DCC/HoBt) method. A five-fold
131
132
ITO AND TACHIBANA
human, porcine, bovine, rat, canine II-IISDAVFTDNYTRLRKQMAVKKYLNS 1
5
10
15
I L N-HH2 20
25
---C-terminal amino a c i d - - G 1 y - -
28
L y s --A (Arg)
r g---
(Lys) Enzymatic Cleavage
FIG. 1. Amino acid sequence of V1P.
Enzymatic Cleavage excess of blocked amino acids and reagents was utilized according to the protocol shown in Fig. 3. Arginine was coupled by the standard DCC method to prevent lactam formation of the N imgroup. His was also coupled by the standard DCC method to prevent destruction of Tos. Coupling was monitored by the standard ninhydrin test (19) and coupling reactions were repeated in an attempt to produce improved coupling when the ninhydrin test proved to be positive. Peptides were acetylated by acetic anhydride if the ninhydrin test failed to demonstrate an improvement after repeating of the coupling reaction. At the completion of synthesis, the N-ct-Boc group was removed with 40% trifluoroacetic acid (TFA) in dichloromethane according to steps 1-9 of the protocol, and the protected peptide resin was dried in vacuo for 18 hours at room temperature. After construction of the desired peptide chain, cleavage of the peptide from the resin and concomitant deprotection were achieved by a single treatment with anhydrous hydrogen fluoride (HF). Protected peptide resin was stirred with HF (20 ml) containing 10% anisole and 5% dimethyl sulfide at - 20°C for one hour. After removal of HF, the residue was washed with diethyl ether by decantation. The crude precipitate was transferred to a glass filter with diethyl ether, washed once more with an excess of diethyl ether, and then dried in vacuo. The mixture of crude products was extracted with 10% acetic acid at room temperature for several minutes, filtered through a fritted funnel, and lyophilized. (A typical chromatogram of crude product is shown in Fig. 4.) At the first purification, the crude peptide was dissolved in 0.05 M ammonium acetate buffer (pH 7.0), loaded into a carboxymethyl-cellulose (CM-C) column (Whatman CM-52, 2.5 x 30 cm), and eluted with a linear 0.05 M to 0.5 M ammonium acetate gradient (pH 7.0). The main fractions were then combined and lyophilized. The second purification was achieved by semipreparative reverse-phase HPLC (RP-HPLC), using a C18 (octadecyl silica; ODS) column (YMC ODS 5 l~m A-343, 20 × 250 mm, Kyoto) with a linear gradient of 20% to 40% acetonitrile (CH3CN) containing 0.01 N hydrochloric acid (HC1) or 1% TFA. Final purification to homogeneity was achieved by elution through a semipreparative HPLC C18 column (YMC ODS A-323 5 i.tm 10 × 250 mm) with a linear gradient of 1% to 45% acetonitrile containing 0.01 N acetic acid (AcOH). The purified peptide was then lyophilized. Homogeneity of the final purified peptides was assessed by analytical RP-HPLC (YMC ODS 5 txm AM-303, 4 . 6 x 250 mm and Nucleosil C18 5 Ixm 4 . 6 × 2 5 0 mm) with a TFA/CH3CN solvent system. The amino acid composition was calculated using a Hitachi amino acid analyzer Model 835 equipped with a system amino acid computing integrator, after the purified product was hydrolyzed with 6 N HC1 containing 1% phenol for 24 hours at 110°C in vacuum tubes. The structure of each synthesized peptide was confirmed by fast atom bombardment (FAB) mass spectrometry performed using a double focusing nmss spectrometer (JOEL, type JMS-HX100, Tokyo). For structural analysis, peptide mapping was performed by digestion with TPCK-treated trypsin (Sigma). Natural VIP and synthetic peptides (10 nmoles) in 50 mM phosphate buffer (100 Ixl, pH 7.4) were incubated together with TPCK-treated trypsin (5 Ixl of a 0.1 mg/ml solution) for 2 hours at 37°C, and then boiled for 5
---C-terminal amino acid- - G I y - - O H Enzymatic Conversion
---C-terminal amino a c i d - - N H 2 FIG. 2. C-terminal amidated peptide.
min to stop the digestion. Thereafter, the solution was directly loaded onto an analytical RP-HPLC column (ODS) to compare it with natural VIP and to confirm the modifications. Typical chromatograms of the fragments are shown in Fig. 6, and each fragment was identified by amino acid analysis.
Biological Assays Bronchodilatory activity. Bronchodilatory activity was determined by the relaxation of isolated guinea pig tracheal smooth muscle. A male guinea pig weighing about 300 g was bled to death from the jugular vein and then thoracotomized. Immediately after the thoracotomy, 3 cm of the trachea was removed and immediately transferred to Krebs solution (NaCI: 118.1 raM, KCI: 4.69 mM, MgSO4:0.60 mM, KH2PO4:0.92 mM, NaHCO3:25 raM, CaC12:2.52 mM, and glucose: 0.2%). This tracheal segment was cut into seven equal-sized rings and these cartilage rings were connected to one another adjacent to the tracheal smooth muscle with cotton yam. Subsequently, the cartilage opposite the smooth muscle was cut, so that the smooth muscle portions could be connected to each other. This preparation was then suspended in an isothermic tissue bath (7 ml). The lower part of the preparation was fixed, while the upper part was connected to an isotonic transducer with a load of 0.5 g, which was used to determine the extent of the relaxation reaction. Krebs solution containing 6.5 x 10 6 M histamine was used to precontract the smooth muscle. This solution was aerated with a mixture of 95% oxygen and 5% carbon dioxide and was maintained at 37°C. The tissues were superfused with the solution at a flow rate of 3.3 ml/min. The relaxation reaction was shown to be proportional to the amount of the test agent that was added. Utilizing this relationship, the relaxation of the tracheal smooth muscle by various synthetic peptides was compared with that achieved by VIP. The VIP standard (10 -5 M) was added in 20 it1 and 40 ILl (200 pmoles and 400 pmoles) or 10 ixl and 20 Ixl aliquots. The peptides under study were also used at 10 -5 M, with the exception that N-terminaldeleted fragments were assayed at 10 - 3 M, and were added as aliquots of 10 Ixl and 20 p.1. The area (height × time) of the relaxation curve for each peptide was determined, and specific activities were obtained by taking the area for VIP as 100% and calculating the relative ratio for the area of each peptide. The relaxation reactions were thus compared by the four-point assay method using the above-mentioned procedure (5,28). Hypotensive effect. The hypotensive effect of each peptide was determined by the changes in carotid artery pressure in a rat. A Sprague-Dawley male rat weighing 200 g was anesthetized by the intraperitoneal administration of 48% urethane at a dose of 2.7
VIP PRECURSORS AND BRONCHODILATION
133
Reagent
Step
Time
CH2C12 wash 3 times 40% TFA/CH2C12 deprotection 40% TFA/CH2C12 deprotection CH2CI2 wash 5 times EtOH wash 3 times CH2C12 wash 5 times 10% TEA/CH2C12 neutralization 10% TEAJCH2CI2 neutralization CH2CI2 wash 5 times Boc-amino acid/CH2C12 delivery 5-fold excess 0.5 M DCC/CH2CI2 delivery 5-fold excess mix for coupling or until monitoring shows complete reaction CH2C12 wash 5 times Recoupling, if necessary, by repeating steps 6-13 CH2CI2 wash 5 times
1:
2: 3: 4: 5: 6: 7: 8: 9: 10: 11: 12: 13: 14: 15:
1 mln 1 man
20 nun 1 nun 1 nun 1 nun 1 nun 10 nun 1 nun
up to 120 min 1 min 1 min
FIG. 3. Protocol for solid-phase peptide synthesis.
ml/kg. The carotid artery was cannulated for the injection of each peptide and a transducer cannula for determining the blood pressure was also inserted into the carotid artery. Peptide samples (12.5 to 400 pmol/kg) were then administered into the carotid artery, and the changes in blood pressure were plotted with a polygraph to obtain dose-response curves. The amount of each sample required to produce a drop of 20 mmHg in blood pressure was determined from the curves. The amount for native VIP was regarded as 100%, and the specific activity of each sample was obtained by calculating the relative ratio.
RESULTS
Peptide Synthesis The crude peptides obtained by cleavage of the protected peptide resins each had a major peak on RP-HPLC analysis (Fig. 4). Synthetic peptides gave a single peak in analytical HPLC after final purification and the comparison of synthetic and natural VIP by analytical RP-HPLC showed that they had the same retention (RT) and eluted together on cochromatography (Fig. 5). The amino acid compositions of these peptides after hydrolysis were consistent with the theoretical values (Table 1). FAB mass spectrometry data also agreed with the theoretical predictions (data not shown). For sequence confirmation peptide mapping, peptides treated with TPCK-trypsin were compared with natural VIP using RP-HPLC, as shown in Fig. 6. The digestion of VIP and synthetic peptides by TPCK-trypsin yielded two C-terminal fragments, as expected from the location of the two lysines (positions
Statistical Methods The parallel line assay was used to estimate the relative potency and 95% confidence limits with the aid of a computer for bronchodilatory activity (13). Comparison of the hypotensive effect was made by A N O V A and Scheffe's multiple comparison.
TABLE 1 AMINO ACID COMPOSITIONS OF VIP AND ITS ANALOGUES AFTER HYDROLYSIS BY 6 N HC1 CONTAINING 1% PHENOL AT 110°C FOR 24 HOURS IN A VACUUM TUBE
Asp
Thr
Ser
Glu
Gly
Ala
Val
Met
Ile
Leu
Tyr
Phe
Lys
His
Arg
Natural VIP Synthetic VIP (theo)
5.0 5.0 (5)
1.9 2.0 (2)
1.8 1.8 (2)
1.1 1.1 (1)
0.2 0 (0)
2.0 2.1 (2)
1.9 2.0 (2)
0.9 1.0 (1)
1.0 1.0 (1)
3.1 3.0 (3)
1.9 2.0 (2)
1.0 1.0 (1)
3.2 3.1 (3)
0.9 1.1 (1)
2.1 2.0 (2)
VIP-OH 1-27-OH 2-28-NH 2 3-28-NH z [Met~7(O)]VIP [Leu17]VIP VIP-G-OH VIP-G-K-OH VIP-G-K-R-OH
5.0 4.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0
1.9 1.9 2.0 1.9 1.9 2.0 1.9 1.9 1.8
1.8 1.7 1.8 0.9 1.7 1.8 1.8 1.8 1.7
1.1 1.1 1.1 1.1 1.1 1.1 1.1 1.1 1.0
0 0 0 0 0 0 1.l 1.1 1.0
2.1 2.0 2.1 2.1 2.0 2.3 2.1 2.1 2.0
2.0 2.0 2.0 2.0 2.0 2.1 2.0 2.0 1.9
0.8 1.0 0.8 1.0 0.6 0 1.0 1.0 1.0
1.0 1.0 1.0 1.0 1.0 0.9 1.1 1.0 0.9
3.1 3.2 3.1 3.1 3.2 4.1 3.1 3.1 3.1
2.0 2.0 2.0 2.0 2.0 2.1 2.0 2.0 2.0
1.0 1.0 1.0 1.0 1.0 1.1 1.0 1.0 1.0
3.0 3.1 3.0 3.0 3.2 3.2 3.0 4.0 4.0
1.1 1.1 0 0 1.1 0.9 1.1 1.1 1.1
2.0 2.0 2.0 2.0 2.0 2.2 2.0 2.0 3.1
One nmole of each peptide was hydrolyzed.
134
ITO AND TACHIBANA
~40
,,~ 0.2<.%
0.4-
A
E C)
-30
cq
~
,~ o,1-
0.3-
I
~(..) z
+
E
g
*zl
F~
-20
4O
3 o.2-
I
10
'~
0.2
z
20
c
u')
~o
3'0
,~o
Time (mln)
8
== ,,,Q
o
FIG. 4. Reverse-phase HPLC analysis of crude VIP-G-K-OH on a C18 column. A linear gradient from 22.5% to 37.5% acetonitrile containing 0.1% TFA was used for 40 min at a flow rate of 1.0 ml/min.
,,D ,,¢
-30
I
,~ 0.1-
1~,
3'0 -40
30
0.1!
20 T i m e (rain)
O
.....
C) I ~0 z
-20
. L~"
1~)
I
Time (min)
20
30 -40
,,m0.2" 20 and 21 in the peptide sequence). Synthetic VIP fragments were revealed to have exactly the same chromatogram as natural VIP by RP-HPLC analysis. C-terminal fragments originating from Cterminal-modified peptides revealed different RTs to the natural fragment, while other fragments were consistent with the same portions of VIP. These tryptic fragments were subjected to amino acid analysis after isolation of each peak by RP-HPLC and hydrolysis with 6 N HC1. Their amino acid compositions were consistent with the expected compositions of the tryptic fragments derived from the peptides (Table 2).
Biological Studies
I
-30
z
o
20
,.Q ,,¢
i 10
20 Time
30
(rain)
ii :)
-40
~0.2-
The potency of the synthetic peptides was evaluated by assessing guinea pig tracheal smooth muscle relaxation in comparison with VIP in vitro and the hypotensive effect in the rat in vivo. Table 3 shows the tracheal relaxation achieved by the synthetic peptides. Among the fragments that were shorter than mature VIP, VIP-OH (which was C-terminal free instead of amidated) had 30% of activity of VIP, and VIP(1-27)-OH (which had deletion of the C-terminal Asn residue) had only 4.5% of the activity of VIP. N-terminal-deleted peptides (2-28 and 3-28) showed under 1% relative potency compared with natural VIP. Among the C-terminal-extended peptides, VIP-Gly-OH showed only 50% of the bronchodilatory activity of VIP, but VIP-Gly-Lys-OH and VIP-Gly-Lys-Arg-OH had, respectively, 210% and 160% of the activity of native VIP. The relative potency of [MetlT(O)]VIP was decreased to one-third of that of the original VIP and [Leu~7]VIP had 70% of the original bronchodilatory activity. Table 4 shows the hypotensive effect on rat blood pressure in vivo. The ED2o mmHg, which means the dose required to reduce the blood pressure of an anesthetized rat by 20 mmHg, was 81 --- 10 pmol/kg for the VIP standard. Relative potency was calculated using this ED2o mmHg value as 100%. The ED2o mmHg for VIP-OH, VIP(1-27)-OH, VIP(2-28)-NH2, and VIP(3-28)-NH 2 was 105---8, 5 1 5 - - 2 6 9 , 937---160, and > 1 0 0 0 pmol/kg (mean--. SEM), respectively. In the case of C-terminal-extended peptides, VIP-Gly-OH, VIP-Gly-Lys-OH, and VIP-Gly-Lys-Arg-
0 "l-
/
,~ o.1
/
u')
t'3
~
30 0.1-
Z
- 20 I 10
I 20 Time
I 30
(rain)
FIG. 5. Reverse-phase HPLC analysis of synthetic and natural VIP on a C18 column. A lineargradientfrom 22.5% to 37.5% acetonitrilecontaining 0.1% TFA was used for 40 min at a flow rate of 1 ml/min. (A) Natural VIP (1 nmole). (B) SyntheticVIP (1 nmole). (C) Cochromatography of natural and synthetic VIP (equal quantities). (D) VIP-G-K-OH (1 nmole). OH had 110--.22, 71---8, and 63---3 pmol/kg. Substitution at 17 position {[MetlT(O)]VIP and [Leu 17 ]VIP} gave potencies of 131-4-47 and 1 0 3 - 14 pmol/kg, respectively. DISCUSSION
The results presented in this study show that the peptides pre-
VIP PRECURSORS AND BRONCHODILATION
135
TABLE 2 AMINO ACID COMPOSITIONS OF VIP-G-K-OHAND TRYPSIN FRAGMENTS AFIT_,RHYDROLYSIS BY 6 N HCl CONTAINING 1% PHENOL AT ll0*C FOR 24 HOURS IN A VACUUMTUBE
VIP-G-K-OH (theo) Fr.1 Fr.2 Fr.3 Fr.4 Fr.5 Fr.6 Fr.7 Fr.8 Fr.9 Fr.10
Asp
Thr
Ser
Glu
Gly
Ala
Val
Met
lie
Leu
Tyr
Phe
Lys
His
Arg
5.0 (5)
1.9 (2)
1.8 (2)
1.1 (1)
1.1 (1)
2.1 (2)
2.0 (2)
1.0 (1)
1.0 (1)
3.1 (3)
2.0 (2)
1.0 (1)
4.0 (4)
1.1 (1)
2.0 (2)
1.0 1.0 1.0 1.0 1.0 1.0 1.0 3.0 2.0 2.0
1.9
0.9 0.8 0.8
0.94 1.08 0.98 0.98 0.90 1.04
1.06 1.23 1.02 1.06 1.05 1.0
0.76 0.85 0.94 0.94 0.90
1.01 0.99
0.99 0.97
1.02 1.03
0.81 2.65 1.69 1.81 1.00 1.29 0.98 1.01 1.00
2.05 2.03
1.00
0.87
1.02
1.83 0.93
One nmole of each peptide was hydrolyzed.
pared by the solid-phase method had the desired sequences and purity. Although the biological activity of a few VIP fragments has been evaluated previously (1, 15, 37), it remained unknown that peptides with the C-terminal free and the almost same size as VIP needed to have a satisfactory biological activity (38). As our purpose was to identify bioactive C-terminal-free VIP, we first tried to shorten the peptide. Therefore, we synthesized the C-terminal amide hydrolyzed peptide VIP-OH and the C-terminal-deleted peptide VIP(1-27)-OH, and compared them with N-terminaldeleted peptides [VIP(2-28) and VIP(3-28)] with respect to their bronchodilatory and hypotensive activities. VIP-OH had almost the same hypotensive activity as the original VIP, whereas its bronchodilatory activity was decreased to one-third of that of VIP. VIP(1-27)-OH maintained one-fifth of the hypotensive activity of the original VIP, whereas its bronchodilatory potency was only
4.5% of that of VIP. As shown in Table 3, bronchodilatory activity was lost from the N-terminal-deleted peptides when the Nterminal His was deleted. On the other hand, their hypotensive activity gradually decreased in proportion to the loss of chain length. In early studies, Bodanszky et al. claimed that some VIP fragments possessed considerable biological activity (1) and Fournier et al. reported fragments with hypotensive activity (15). Our results in regard to hypotensive activity support their find-
ot
50
i
25
0.1
0
o[
TABLE 3 RELATIVE POTENCY OF GUINEA PIG TRACHEAL RELAXANT ACTIVITY IN VITRO
B
I
50
Bronchodilatory Activity Synthetic VIP A) VIP-OH VIP(1-27)-OH VIP(2-28)-NH2 VIP(3-28)-NH2
25
Defined as 100%
o
30% (5-35%)* 4.5% (0.4--5.5%)* < 1.0% < 1.0%
n= 8 n= 8 n= 8 n= 8
VIP-G-OH VIP-G-K-OH VIP-G-K-R-OH
50% (40-60%)* 210% (190-240%)* 160% (140--190%)*
n = 16 n = 16 n= 8
C) [MetI7(O)]VIP [Leu~7]VIP
30% (20-40%)* 70% (65-85%)*
n= 8 n= 8
B)
"~
0.1 .
.
.
.
.
.
.
.
.
.
" "
25
o.
A) Comparison of fragments with C- and N-terminal deletions. B) Comparison of precursors. C) Comparison of substitution at position 17. *95% confidence limits.
~o
o
0
10
20 Tlme (rnln)
30
FIG. 6. Reverse-phase HPLC analysis of trypsin-digested fragments of VIP and its analogues on a C18 column. Each peptide (10 nmoles) was digested with 20 p,g of trypsin (TPCK-treated) in 100 p,1 of 0.1 M phosphate buffer (pH 7.4) at 37°C for 6 h. Half of the reaction mixture was loaded directly into the column, and separated using a linear gradient of 1% to 48% acetonitrile containing 0.1% TFA for 40 rain at flow rate of 1.0 ml/min. (A) natural VIP. (B) synthetic VIP. (C) VIP-Gly-Lys-OH.
136
ITO AND TACHIBANA
TABLE 4 COMPARISON OF HYPOTENSIVE ACTIVITY IN VIVO
Synthetic VIP A) VIP-OH VIP(1-27)-OH VIP(2-28)-NH 2 VIP(3-28)-NH 2
ED2o mmHg* (pmol/kg) (n_-----8)
Relative PotencyS-
81 ~ 10
100%
105 515 937 >
+ 8 -+ 269§ --- 160§ 10000
80% 20% 10% < 1%
B) VIP-G-OH VIP-G-K-OH VIP-G-K-R-OH
110 - 22 71 --- 8:~ 63 _+ 3:~
70% 110% 130%
C) [MetlT(o)]vIP [LeuaT]VIP
131 --- 47 103 --- 14
60% 80%
A) Effect of fragments with C- and N-terminal deletions. B) Effect of precursors. C) Effect of substitutions at position 17. *ED2o mmHg means the dose of the sample required to produce a drop of 20 mmHg in the blood pressure of an anesthetized rat. Values are the means ---SE. tVIP as 100%. ~p<0.05, §p<0.01.
ings, but our data on bronchodilatory activity are inconsistent with previous observations. Our results suggest that the essential structures for producing bronchodilation were more strictly limited than those related to the hypotensive effect of VIP. Although these findings confirmed that there should be no smaller C-terminalfree peptides than VIP itself, we considered that there may be active forms of C-terminal-free VIP because VIP-OH had a comparable activity to natural VIP. Accordingly, we synthesized several C-terminal-extended precursors of VIP: VIP-Gly-OH, VIPGly-Lys-OH, and VIP-Gly-Lys-Arg-OH. The latter two synthetic
peptides showed an unexpectedly high bronchodilatory activity (Table 3). On the other hand, the precursor VIP-GIy-OH, which represents VIP just before maturation, had only about half the potency of VIP in bronchorelaxant assays, while displaying a hypotensive activity equal to 80% of that of mature VIP. These resuits raised an interesting question as to whether bioactive Cterminal-extended precursors of VIP play a role in mammalian physiology. It is quite unusual for precursors to possess higher bioactivities than the mature form of a peptide. Recently, C-terminal-extended secretin precursors (secretin-Gly-OH and secretin-Gly-Lys-Arg-OH) were isolated from porcine upper intestinal tissue, and the latter peptide exhibited a substantially higher bioactivity than secretin itself (16). In the case of VIP, other reports also support our findings and it may be that variations in the structure of VIP led to its tissue selectivity (12, 13, 17, 31, 38). As secretin and VIP belong to the same family of gastrointestinal hormones, there may be a similar reason for the existence of highly active precursors that is at present unknown. It has been reported that the Met 17 residue of VIP is easily oxidized to produce [MetIT(o)]vIP (37). However, there has been no quantitative and comparative data available on the bioactivities of these peptides. We synthesized the oxidized form Met17(O) by the solid-phase method, using Boc-Met(O) instead of Boc-Met. The presence of methionine sulfoxide in the peptide sequence was confirmed by HPLC, which distinguished the oxidized form from VIP (results not shown). The bronchodilatory activity of the oxidized peptide was decreased to one-third of that of natural VIP, but the hypotensive potency only decreased slightly. This finding was in good accord with the previously mentioned suggestion that bronchodilation seems to demand a strict structure while the structure producing the hypotensive effect is permissive. [Leu~7]VIP had a potency nearly equivalent to VIP itself for both bronchodilatory and hypotensive activity. PHI and PHM, which are closely related in sequence and biological properties to VIP, have Leu in position 17 of their sequences instead of the Met ~7 of VIP and are encoded by the same DNA (18, 32, 33). Furthermore, it was reported recently that helodermin, which is a C-terminal extended VIP-like peptide and has Leu 17 in its sequence, also had bronchodilatory properties (13). These and our findings suggest that C-terminal-free VIP with Leu 17 may have a high level of both bronchodilatory and hypotensive activity.
REFERENCES 1. Bodanszky, M.; Henes, J. B.; Yiotakis, A. E.; Said, S. I. Synthesis and pharmacological properties of the N-terminal decapeptide of the vasoactive intestinal peptide (VIP). J. Med. Chem. 20(11):14611464; 1977. 2. Bodanszky, M.; Bodanszky, A.; Said, S. I. Synthesis of the avian vasoactive intestinal peptide (VIP). Fed. Proc. 37:829; 1978. 3. Carlquist, M.; Mutt, V.; Jornvall, H. Isolation and characterization of bovine vasoactive intestinal peptide (VIP). FEBS Lett. 108(2): 457-460; 1979. 4. Carlquist, M.; McDonald, T. J.; Go, V. L. W.; Bataille, D.; Johansson, C.; Mutt, V. Isolation and amino acid composition of human vasoactive intestinal polypeptide (VIP). Horm. Metab. Res. 14:2829; 1982. 5. Castillo, D. B. The tracheal chain 1. A preparation for the study of antispasmodics with particular reference to bronchodilator drugs. J. Pharrnacol. Exp. Ther. 90:104-109; 1947. 6. Colombo, R. A new solid-phase synthesis of porcine vasoactive intestinal peptide using Na-9-fluorenylmethyloxycarbonyl amino acids. Experientia 38:773-775; 1982. 7. Coy, D. H.; Gardner, J. Solid-phase synthesis of porcine vasoactive intestinal peptide. Int. J. Pept. Protein Res. 15:73-78; 1980. 8. Dey, R. D.; Shannon, W. A., Jr.; Said, S. I. Localization of VIPimmunoreactive nerves in airways and pulmonary vessels of dogs,
cats, and human subjects. Cell Tissue Res. 220:231-238; 1981. 9. Dimaline, R.; Reeve, J. R., Jr.; Shivelly, J. E.; Hawke, D. Isolation and characterization of rat vasoactive intestinal peptide. Peptides 5: 183-187; 1984. 10. Du, B. H.; Eng, J.; Hulmes, J. D.; Chang, M.; Pan, Y. C. E.; Yalow, R. S. Guinea pig has unique mammalian VIP. Biochem. Biophys. Res. Commun. 128(3):1093-1098; 1985. 11. Eng, J.; Du, B.-H.; Raufman, J.-P.; Yalow, R. S. Purification and amino acid sequences of dog, goat and guinea pig VIPs. Peptides 7(Suppl. 1):17-20; 1986. 12. Fahrenkrug, J.; Ottesen, B.; Palle, C. Non-amidated forms of VIP (glycine-extended VIP and VIP-free acid) have full bioactivity on smooth muscle. Regul. Pept. 26:235-239; 1989. 13. Finney, D. J. Statistical method in biological assay (2nd edition). New York: Hafner Publishing Co.; 1964. 14. Foda, H. D.; Said, S. I. Helodermin, a C-terminal extended VIP-like peptide, evokes long-lasting tracheal relaxation. Biomed. Res. 10(1): 107-110; 1989. 15. Fournier, A.; Saunders, J. K.; St-Pierre, S. Synthesis, conformational studies and biological activities of VIP and related fragments. Peptides 5:169-177; 1984. 16. Gafvelin, G.; Carlquist, M.; Mutt, V. A proform of secretin with high secretin-like bioactivity. FEBS Lett. 184(2):347-352; 1985.
VIP PRECURSORS AND BRONCHODILATION
17. Gafvelin, G.; Andersson, M.; Dimaline, R.; Jornvall, H.; Mutt, V. Isolation and characterization of a variant form of vasoactive intestinal polypeptide. Pcptides 9:469--474; 1988. 18. Itoh, N.; Obata, K.; Yanaihara, N.; Okamoto, H. Human preprovasoactive intestinal polypeptide contains a novel PHI-27-1ike peptide, PHM-27. Nature 304(11):547-549; 1983. 19. Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Color test for detection of free terminal amino groups in the solid-phase synthesis of peptide. Anal. Biochem. 34:595-598; 1970. 20. Larsson, L.-I.; Fahrenkrug, J.; Schafflitzky de Muckadell, O.; Sundler, F.; Hakaqnson, R.; Rehfeld, J. Localization of vasoactive intestinal polypeptide (VIP) to central and peripheral neurons. Proc. Natl. Acad. Sci. USA 73:3197-3200; 1976. 21. Leys, K.; Morice, A. H.; Madonna, O.; Sever, P. S. Autoradiographic localisation of VIP receptor in human lung. FEBS Lett. 199(2): 198-202; 1986. 22. Lundberg, J. M.; Fahrenkrug, J.; Hokfelt, T.; Martling, C. R.; Larsson, O.; Tatemoto, K.; Anggard, A. Co-existence of peptide HI (PHI) and VIP in nerves regulating blood flow and bronchial smooth muscle tone in various mammals including man. Peptides 5:593-606; 1984. 23. Matsuzaki, Y.; Hamasaki, Y.; Said, S. I. Vasoactive intestinal peptide: A possible transmitter of non-adrenergic relaxation of guinea pig airways. Science 210:1252-1253; 1980. 24. Mutt, V.; Said, S. I. Structure of the porcine vasoactive intestinal octacosapeptide. Eur, J. Biochem. 42:581-589; 1974. 25. Nilsson, A. Isolation, amino acid composition and terminal amino acid residues of the vasoactive octacosapeptide from chicken intestine. Partial purification of chicken secretin. FEBS Lett. 47:284-289; 1974. 26. Nishizawa, N.; Hayakawa, Y.; Yanaihara, N.; Okamoto, H. Nucleotide sequence divergence and functional constraint in VIP precursor mRNA evolution between human and rat. FEBS Lett. 183:55-59; 1985. 27. O'Donnell, M.; Garippa, R.; Welto, A. F. Relaxant activity of atriopeptins in isolated guinea pig airway and vascular smooth muscle. Peptides 6:597-601; 1985. 28. Perry, W. L. M. Pharmacological experiments on isolated prepara-
137
tions. London: Livingstone Ltd.; 1968. 29. Polak, J. M.; Bloom, S. R. Localization and measurement of VIP in the genitourinary system of man and animals. Peptides 5:225-230; 1984. 30. Said, S. I.; Mutt, V. Isolation from porcine intestinal wall of vasoactive octacosapeptide related to secretin and glucagon. Eur. J. Biochem. 28:199-204; 1972. 31. Simoncsits, A.; Tjomhammar, M. L.; Kalman, M.; Cserpan, I.; Gafvelin, G.; Bartfai, T. Synthesis, cloning and expression in Escherichia coli of artificial genes coding for biologically active elongated precursors of the vasoactive intestinal polypeptide. Eur. J. Biochem. 178:343-350; 1988. 32. Suzuki, S.; Araki, K.; Tachibana, S. Isolation of porcine vasoactive intestinal polypeptide and comparison of its biological activities with secretin and glucagon. Yakugaku Zasshi 99:172-179; 1979. 33. Tachibana, S.; Ito, O. Novel bioactive precursor analogues of vasoactive intestinal polypeptide (VIP)--Structure-activity relationships. In: Shiba, T.; Sakakibara, S., eds. Peptide Chemistry 1987. Osaka: Protein Research Foundation; 1988:481--486. 34. Tatemoto, K.; Mutt, V. Isolation and characterization of the intestinal peptide porcine PHI (PHI-27), a new member of the glucagonsecretin family. Proc. Natl. Acad. Sci. USA 78(11):6603--6607; 1981. 35. Tatemoto, K.; Jonvall, H.; McDonald, T. J.; Carlquist, M.; Go, V. L. W.; Johansson, C.; Mutt, V. Isolation and primary structure of human PHI (peptide HI). FEBS Lett. 174(2):258-261; 1984. 36. Wang, S. C.; Du, B. H.; Eng, J.; Chang, M.; Hulmes, J. D.; Pan, Y.-C. E.; Yalow, R. S. Purification of dog VIP from a single animal. Life Sci. 37:979-983; 1985. 37. Yanaihara, N.; Nokihara, K.; Yamamoto, K.; Hashimoto, T.; Yanaihara, T.; Ito, Z.; Yamaguchi, K.; Abe, K. Immunological aspects of secretin, substance P and VIP. Gastroenterology 72:803810; 1977. 38. Yiangou, Y.; Marzo, V. D.; Spokes, R. A.; Panico, M.; Morris, H. R.; Bloom, S. R. Isolation, characterization, and pharmacological actions of peptide histidine valine 42, a novel prepro-vasoactive intestinal peptide-derived peptide. J. Biol. Chem. 262:14010-14013; 1988.