- Email: [email protected]
A variety of Mytilus inhibitory peptides in the abrm of Mytilus edulis: Isolation and characterization
0306~4492/91 $3.00+ 0.00 0 1991Pergamon press plc
Camp. Biochem. Physiol.Vol. IOOC,No. 3, pp. 525-531, 1991
Printed in Great
Britain
A VARIETY THE ABRM
OF MYTKUS INHIBITORY PEPTIDES IN OF MYTKUS EDULIS: ISOLATION AND CHARACTERIZATION
YUKO FumAwA,*‘f ICHIRO KUBOTA,~ TETSUYAIEEDA, HIROYIJKI MINAKATA$ and YOJIRO MUNEOKA *Faculty of Integrated Arts and Sciences, Hiroshima University, Hiroshima 730, Japan; $Suntory Bio-Pharma Tech Center, Chiyoda-machi, Oura-gun, Gunma 370-05, Japan; and @untory Institute for Bioorganic Research, Shimamoto-cho, Mishimagun, Osaka 618, Japan (Received 21 December 1990)
Abstract-l. Five species of Mytilus inhibitory peptides, MIP,_,, were isolated from acetone extracts of the anterior byssus retractor muscle (ABRM) of Mytilus edulis. MIP, and MIP, were shown to be Sr-MIP and A2-MIP, respectively, first isolated from the pedal ganglia of the animal. 2. All the five peptides had a common C-terminal structure of -Pro-Xaa-Phe-Val-NH,, which was shown to be important for their biological activity. 3. The five MIPS showed similar inhibitory effects on contractions of the ABRM but did not affect catch tension and its relaxation. 4. In addition to the MIPS, catch-relaxing peptide (CARP) was also found in the ABRM.
INTRODUCTION In 1987 and 1988, three neuropeptides
MATERIALS AND were isolated
from the pedal ganglia of the bivalve mollusc Mytifus edulis (Hirata et al., 1987, 1988). One was termed “catch-relaxing peptide” (CARP) whose structure was determined to be Ala-Met-Pro-Met-Leu-ArgLeu-NH, . CARP showed a potent relaxing effect on catch tension of the anterior byssus retractor muscle (ABRM) of Mytilus. The other two were homologous peptides termed Mytilus inhibitory peptides (MIPS) after their remarkable inhibitory actions on contractions of the ABRM. Their structures were determined to be Gly-Ser-Pro-Met-Phe-Val-NH, (S*-MIP) and Gly-Ala-Pro-Met-Phe-Val-NH, (A*-MIP). Since the pedal ganglion is the main origin of the nerves innervating the ABRM (for review, see Muneoka et al., 1990), we expected that the peptides in the ganglion should be transported to, and stored in, the nerve terminals in the ABRM, to be released during neural excitation. Based on this expectation, we have attempted to isolate bioactive peptides from the ABRM. So far, we have found CARP, the two MIPS and also a number of other peptides including novel MIP-related peptides. At several conferences, we have presented their possible structures and representative actions (Fujisawa et al., 1990a, 1990b), but we have not yet published complete structure and detailed pharmacological and physiological characteristics of any of the peptides. Here we report the structures and actions of five species of MIPS and CARP isolated from the ABRM.
tTo whom correspondence should be addressed.
METHODS
Animals Mytilus edulis were collected from Hiroshima Bay and stored in a tank filled with aerated artificial seawater (ASW) at 17-24°C. For physiological experiments, animals were used within 7 days of collection, and for peptide purification, those within 10 hr were used. Meretrix lusoriu were purchased from a commercial source and stored in ASW. Euhadra congenita hiconis were collected at the campus of Hiroshima University. They were kept in the laboratory at 18-25°C and fed on sweet potatoes. Recordings of muscle activity
The isolated muscle preparation was suspended in a 1.5 ml polypropylene chamber. One end of the muscle was tied with a cotton thread and connected to a force transducer. The methods of stimulation and recording of contractions of the ABRM were essentially the same as those of Muneoka and Twarog (1977). The methods of recording of spontaneous contractions of the heart of Meretrix and the crop of Euhadra were essentially the same as those of Hirata et al. (1989) and Kuroki er al., respectively. Peptide purification
The ABRMs excised from 10,000 animals were homogenized in 100% and then 80% acetone (0.25 g tissue wet wtjml). The extracts were concentrated by a rotary evaporator, dissolved in 0.1 M HCI and forced through C,, cartridges (Waters, Sep-Pak). The retained material, eluted with 100% methanol, was applied to a Sephadex G-15 column (26 x 400mm) and eluted with 0.1 M acetic acid. Bioactivity of an aliquot from each fraction (4ml) was assayed on phasic contraction elicited by repetitive electrical pulses of stimulation (15 V, 3 msec, 10 Hz, for 5 set) and on catch tension produced by application of 10-“M acetylcholine (ACh) in the ABRM preparation. The gel-filtrated fractions which showed an activity on the ABRM were pooled and concentrated under reduced pressure. The concentrated material was injected into a C,, 525
YUKO
526
FUSAWA
Fig, 1. Anion-exchange HPLC profile of the second step of pu~~~~on of the ABRM extracts. Fractions indicated by A and B showed an inhibitory effect on phasic contraction of the ABRM. HPLC column (TSKgeI ODS-80T,, 4.6 x I50 mm, Tosoh) and eluted with a 60-min linear gradient of o-60% acetonitrile in 0.1% trifluornacetic acid (TFA) at pH 2.2. The active fractions from which the peptides reported here were purified were then injected into an con-exchange column frSKge1 DEAE-5PW, 7.5 x 75 mm, Tosoh) and elutedwith 10 mM T&ma-base at pH 9.6. The flowthrough fractions and the fractions eluted between 25 min and 35 min showed an inhibitory action on phasic contraction of the ABRM (Fig. 1). The tIow-through fractions (indicated by A in Fig. I) were injected into a cation-exchange cohmm (TSKgel SF-SPW, 7.5 x 75 mm, Tosoh) and eluted with a 58-min linear gradient of o-O.5 M NaCl in 20 mM phosphate buffer at pH 6.7. Several peaks of bioactivities were eluted at different positions. In the present experiments, further purification was carried out for peak I, II and III (Fig. 2). The fractions of each peak were separately applied to a C8 column (Finepak SIL CsS, 4.6 x 25Omm, Jasco) and eluted with a 40-min linear gradient of 2040% acetonitrile in 0.1% TFA. MIP, and MIPX were purified from peak I by two more steps of reversed-phase HPLC. CARP was purified from peak II by another step of reversed-phase HPLC. MIP, and MIP, were purified from peak III by one and two more steps of reversed-phase HPLC, respectively. The other group of inhibitory fractions obtained in the anion-exchange HPLC (indicated by B in Fig. I) was applied to the C, cc&mu and eluted with a 2&min linear gradient of 2025% acetonitrile in 0.1% TFA at pH 2.2 The inhibitory fractions were then applied to the C,, column and eluted isocratically with 20% acetonitrile in 0.1% TFA. MIPj was thus purified.
et al.
0
20
%Q
90 Tii
1~~
Fig. 2. ~a~on~x~h~~e HPLC profile of the next step of purification of fractions A in Fig, 1. Fractions I and III inhibited phasic contraction of the ABRM while fractions II potentiated it and relaxed catch tension caused by ACh. The HPLC charts of the &al purification for all the peptides are shown in Fig. 3.
All the purified substances underwent amino acid sequence analysis by the automated Edman degradation method with a gas-phase sequencer (Applied Biosystem 470A). Quantitative amino acid analysis was also performed for all the peptides. In addition, fast atom ~mbar~ent mass spectrometric (FAB-MS) measurement was carried out for MIP,, MIP, and MIFs, which had been supposed to be novel substances. Canjrmation of the proposed structures In order to confirm the structures proposed in the analyses, we compared the chemical and biological characteristics of each of the synthesized peptides to those of the native one. First, the behavior on the reversed-phase and the ion-exchange HFLC was compared. Secondly, the relation between doses and effects was compared in a ABRM preparation. Saline and drugs
The physiological saIine for the ABRM of ~~r~~~ and the heart of &ferer&x was ASW whose ~om~sition was: 445mM NaCl, IOmM KCl, 10mM Cat&, 55 mM MgCl, and 10 mM Tris-HCl (PH 7.8). The composition of the physiological saline far the land snail Euhadra was: 51 mM NaCl, 3.7mM KCl, 10mM CaCl,, 12.6mM MgClz, c
010
oTime
-
0
lo
(mini
Fig. 3. Reversed-phase HPLC profiles of the final step af purification, Arrows indicate u.v.-absorbant peaks of the peptides. a, MIP, ; b, MIP,; c, MIP,; d, MIP,; e, MIP,; f, CARP. The columns were TSKgeI ORS-SOT, (a-c) and Finepak SIL G5 (d-f). Each peptide was eluted isocratically with 0.1% TFA containing different concentrations of acetonitrile (a and b, 18%; c, 20%; d, 22%; e, 22%; f, 28%).
521
Mytilus inhibitory peptides in the ABRM 1.5 mM glucose and 10mM HEPESNaOH (PH 7.5). Acetylcholine bromide (ACh) was purchased from Sigma, and FMRFamidd, from Peninsula Labs Inc. CARP. MIP, (S2-MIP). MIP, (A2-MIP) and MIP fratzrnents (PMFVamidk‘and MFVamide) were synthesized byDr N. Iwasawa at the Suntory Institute for Biomedical Research (Osaka, Japan). Another MIP fragment, FVamide, was synthesized by Dr. H. Minakata at the Suntory Institute for Bioorganic Research. MIP,, MIP,, and MIP, were synthesized at the Peptide Institute (Osaka, Japan). Their structures were confirmed by the sequence analysis and the amino acid analysis. RESULTS
Structures of peptides The results of sequence analysis, amino acid analysis and FAB-MS were shown in Table 1. For MIP, , MIP, and MIP, , the C-terminal residue Val was not detected in the sequence analysis but the results of the amino acid analysis showed the presence of Val. The results of FAB-MS also suggested that these peptides had -Val-NH, at their C-terminal portions. Thus, we predicted the structures of the purified peptides as follows: MIP, (S2-MIP): Gly-Ser-Pro-Met-Phe-Val-NH, MIP, (A2-MIP): Gly-Ala-Pro-Met-Phe-Val-NH, MIP,: Asp-Ser-Pro-Leu-Phe-Val-NH, MIP,: Tyr-Ala-Pro-Arg-Phe-Val-NH, MIP,: Ala-Ser-His-Ile-Pro-Arg-Phe-Val-NH, CARP: Ala-Met-Pro-Met-Leu-Arg-Leu-NH, Each of the synthetic peptides with these structures showed the same profiles as those of the native one on the reversed-phase and the ion-exchange HPLC (Fig. 4). The bioactivity of the two peptides on the ABRM also coincided (Fig. 5). Thus, we concluded that the proposed structures are correct. The newly identified peptides, which were apparently homologous to S2-MIP and A*-MIP, were termed MIP,, MIP4 and MIP,, respectively, and S2-MIP and A2MIP were renamed MIP, and MIP,, respectively. It is notable that these five MIPS have in common Pro-Xaa-Phe-Val-NH, at their C-terminal parts.
Table 1. Results of structural Peptide MIP, MIP, MIP,
MIP,
MIP,
CARP
Effects of MIPS on the ABRM It has been shown that MIP, and MIP, inhibit contractions of the ABRM of Mytilus, e.g. phasic contraction caused by repetitive electrical pulses and tonic contractions caused by ACh and FMRFamide, and that the two peptides are almost equipotent in inhibiting these contractions (Hirata et al., 1988, 1989). In the present experiments, the newly isolated MIPS (MIP,, MIP, and MIP,) also inhibited the phasic contraction, ACh contraction and FMRFamide contraction (Fig. 6). The order of potency of the inhibitory effect on phasic contraction was MIP, > MIP, > MIP, > MIP,. In the case of ACh contraction, too, MIP, was more potent than MIP,, MIP, and MIP,, though MIP, was less effective than MIP, and MIP, In the case of FMRFamide contraction, the order of potency depended on preparations and significant difference in potency between the peptides was not observed. In some preparations, the FMRFamide contraction, after washing out MIPS, was more depressed than that recorded in the presence of the peptides (Fig. 6D). It has been also reported that MIP, and MIP, do not affect catchrelaxing response (Hirata et al., 1989). As well as these peptides, the three new MIPS neither relaxed catch tension nor changed relaxing response to repetitive electrical pulses of stimulation (Fig. 7). All the five members of MIP family conserve Pro-Xaa-Phe-Val-NH, at their C-terminal parts. We have shown that the C-terminal tetrapeptide (ProMet-Phe-Val-NH,) and even the tripeptide (Met-PheVal-NH,) of MIP, and MIP, can inhibit phasic contraction of the ABRM, although the effects are weaker than those of the native hexapeptides (Fujisawa et al., 1990b). In the present experiments, we confirmed these findings and also observed that even the dipeptide fragment, Phe-Val-NH,, which is common to all the five MIPS, showed an inhibitory effect on phasic contraction (Fig. 8). It is notable that the deletion of Pro residue from the tetrapeptide markedly reduced the inhibitory potency. Effects of MIPS on other molluscan muscles MIPS showed inhibitory effects not only on the ABRM but also on other molluscan muscles. They
analyses of the purified
peptides
Results of analyses + ) (I) Gly( 15O)-Ser(37)-Pro(35)-Met(4)-Phe(2)-Val( (2) Ser(l.03), GIy(1.07), Val(l.Ol), Met(O.‘ll), Phe(l), Pro(0.95) (I) Gly(l31)-Ala(lO3)-Pro(23)-Met(4)-Phe(3)-Val(+) (2) Gly(l.lS), Ala(1.09). Va1(1.04), Met(0.76). Phe(l), Pro(0.96) (1) Asp( I SO)-Ser(22)-Pro( 17)-Leu(S)-Phe(3) (2) Asp(1 .oO), Ser(0.91), Pro(0.93). Val(0.97), La(l), Phe(0.99) (3) 676.2 (676.4) (1) Tyr(l7)-Ala(21)-Pro(3l)-Arg(2)-Phe(9) (2) Ala(l.03). Vd(l), Tyr(0.92), Phe(0.96), Arg(1.33). Pro(0.96) (31 751.4 (751.5) ‘-’ &(146)-&( (I) 1 I)-His(4)-Ile(l9)-Pro(22)-Arg(7)-Phe(2) (2) Ser(0.94), Ala(0.96), Val(0.94), Ile(0.93), Phe(l), His(O.SS), Arg(l.04). (3) 925.3 (925.5) (1) Ala(444)-Met(262)-Pro(l27)-Met(l35)-Leu(l lO)-Arg(14)-Leu(33) (2) Ala(l.OE), Met(1.37), Leu(2). Arg(1.25), Pro(+)
Pro(0.78)
(1) Amino acid sequence analysis. Each value means yield of PTH-amino acids (pmol). (2) Amino acid analysis. Each value means relative amount of amino acids normalized on the underlined amino acid indicated by italic. (3) FAB-MS measurement. The values are m/z of molecular ion peaks, indicating molecular weight plus one for H+. Theoretical value for each peptide (amidated form) was shown in ( ).
528
YUKO FUJISAWA et al.
.008
r
1
CARP
Fig. 4. Comparison of retention time on reversed-phase HPLC between native and synthesized peptides. Each of the native peptides (N, upper trace) and the synthetic peptide (S, middle trace) were eluted at the same retention time. The mixture of the two was co-eluted as a single u.v.-absorbant peak (N + S, lower trace). Similar results were obtained on ion-exchange HPLC (not shown). loo-
100
MlP*
. . / ./ .
2 gso._
:/
iG = 5
OL
8
3x1o’8
3x10-9
10-4 Concentration (MI
/
I
o-,
1
10-Q
MIPJ 1
3x10’0
10-P
I 3x10-4
concentmtii
10-a
(MI
oL, 3xlo-s
1
lo-a
#
3x10-8
Concentratiin (M)
50MIP,
I
6
MIPS
-1 d .
7 /
s 3 30 g
25/
B
./’ . OL, 3x10.9
I
10-a
I
I
3x10-4
concentmtii
(M)
10”
/
0
<,
10-s
,(
3x10-’ 3x10-s 10-7 Concentmtion (M)
Fig. 5. Comparison of bioactivities on the ABRM between native and synthesized peptides. The inhibitory effects of MIPS were examined on phasic contraction. Decrease in peak tension was expressed by per cent of the control peak tension. The relaxing activity of CARP was examined on catch tension caused by 10e4 M ACh. Decrease in catch tension during exposure to CARP for 4 min was expressed by percent of the tension just before applying CARP. 0, Native peptides; a, synthesized peptides.
I
10”
Mytilus inhibitory peptides in the ABRM
529
Fig. 6. Inhibitory effects of MIPS on the ABRM. A: effects on phasic contraction elicited by repetitive electrical pulses. The electrical stimulation was applied every 10 min. MIPS were applied 8 min prior to the contractions (upward arrows) and washed out soon after them (downward arrows). B: effects on ACh contraction. ACh 10e5 M was applied for 2 min every 20 min. MIPS were applied 10 min prior to the ACh contractions (upward arrows) and washed out soon after them (downward arrows). After each recording, catch tension was relaxed by repetitive electrical pulses. C: effects on 10e6 M FMRFamide contraction. Experimental procedure was similar to that in B. D: an example of the “outlasted inhibitory effect” of MIPS. Note that the peak tension after washing out MIP is smaller than that during exposure to the peptide.
inhibited the cardiac activity of the bivalve Meretrix Iusoria (Fig. 9A) and the spontaneous activity of the crop of the pulmonate Euhadra congenita hiconis (Fig. 9B). In the heart of Meterix, inhibitory effects of MIPS considerably varied with preparations. In each of the animals, the five peptides showed qualitatively similar inhibitory effects, though they were not equipotent. MIP, was the most potent both in Meretrix and in Euhadra; it markedly and in some
Fig. 7. Effects of MIPS on relaxation of the ABRM by repetitive electrical pulses (15 V, 3 msec, 1 Hz, for 10 set). MIPS did not cause relaxation of catch by themselves and did not affect catch-relaxing response to electrical stimulation (ES).
preparations completely depressed the spontaneous activities of the heart and the crop at low doses such as lo-* to lo-‘M. DISCUSSION
The ABRM of Mytilus was shown to contain CARP and five species of MIPS. These peptides are supposed to be present in the nerve elements in the ABRM and to act as physiological neuromediators. Especially, the presence of CARP, MIP, and MIP,, both in the pedal ganglion and in the ABRM, strongly supports the notion that they are the neuropeptides synthesized in the ganglionic cell body, transported to the nerve terminals and released to the muscle fibers during neural excitation. The origin of the other three MIPS have not been clarified yet but it is possible that they are also contained in the pedal ganglion. In order to establish physiological roles of these peptides, further experiments including electrophysiological and histochemical ones are required. All the five MIPS isolated from the ABRM are closely related to each other both in their structure and biological activity. They exhibit qualitatively similar effects on the ABRM; that is, they exclusively inhibit the contractions and do not affect the
530
YUKOFUJISAWA et al.
~&~~~~/Q 5
PYFVa
MIPZ
MFVa
MFVa
M
:“;;nm
FVa
Fig. 8. Inhibitory effectsof C-terminal fragments of MIPs on phasic contraction of the ABRM in response to repetitive electrical pulses of stimulation. Note that the deletion of Pro from PMFVamide markedly decreases the inhibitory potency. relaxation of catch. The structure common to the five peptides was found to be important for these inhibitory effects; the C-terminal dipeptide Phe-Val-NH, is thought to be essential, and another common residue Pro, which is situated at the forth position from the C terminus, seems to play an important role. These findings may suggest that the five MIPS interact with the same class of receptors which requires the tetrapeptide sequence. Pro-Xaa-Phe-Val-NH,. The other residues, which seem not to be crucial to receptor-ligand interaction, may cause the quantitative difference in inhibitory potency of the five MIPS; they affect the ability of peptide molecules to bind the receptors and the resistance to degradation enzymes. Thus, it can be supposed that the structural variety in MIPS have little physiological importance and that the five analogous MIPS are recognized as the same kind of molecules within the MIP-operating system. If this is true, the MIP system could offer an hypothesis concerning the evolution of signal molecules; when a variation was produced from an ancestral form by gene duplication and mutation, it could be conserved unless the change in the molecule severely affects the ability to bind its receptors. Whether this is true or not, a great deal of information are required A. Meretrix
to answer the question why such many analogs are involved in the regulation of a muscle. It has been proposed that MIP systems are distributed in other molluscs since MIP, and/or MIP, have inhibitory effects on the heart beat of the bivalve, Tapes japonica, twitch and tetanic contractions of the penis retractor muscle of the pulmonate, Achatinafulica (Hirata er al., 1988, 1989) and also on the identified neurones of Achatina (Yongsiri et al., 1989) and of Helix pomatia (Kiss, 1990). In the present experiments, the five members of MIPS showed similar effects on each of Meterix and Euhadra, suggesting that these animals may also have a class of receptors with which a variety of MIP-like peptides interacts, as in the case of Mytilus. Recently, Ikeda et al. isolated as many as 13 MIP analogs from the central nervous system of Helix by using the ABRM as a bioassay system (Ikeda et al., 1991). They have -Pro-Xaa-Phe-Val-NH, except one peptide with -Pro-Xaa-Phe-Ile-NH,. In a prosobranch mollusc Fusinus ferrugineus, however, MIPS of Mytilus showed little or no effect on contractions of the radula retractor muscle and the proboscis retractor muscle (unpublished). In addition, we have failed to isolate MIP-like peptides from the acetone extracts of
heart
MIP3
IO-’ M MIP, 0.59 lmin
MlPl 0. Euhadra crop
IO-’ M MIPI
MlP2
1
MIP3
MIPS Fig. 9. Effects of MIPS on other molluscan muscles. A: effects on the heart beat of Meretrix. B: effects on the spontaneous activity of the crop of Euhadra.
Mytilus inhibitory peptides in the ABRM the central ganglion masses of Fusinus by using the radula muscle as a bioassay system. From a view point of phylogeny, however, prosobranchia and opisthobranchia can be expected to have MIP-related peptides as well as bivalvia and pulmonata. Study on the distribution of MIP-related peptides in other classes of molluscs should be done by using the ABRM as a bioassay system. In addition to CARP and the five MIPS, we have found many other bioactive peptides including at least two MIP-like peptides (unpublished) in the ABRM, though their structures have not been completely confirmed yet (Fujisawa et al., 1990a,b). The ABRM of Mytilus might be a good example of the muscles which are regulated by a number of neuropeptides as well as by the classical neurotransmitters such as ACh or S-HT. Involvement of multiple analogous peptides which seem to exert the same kind of action might be one of the interesting phenomena in such a muscle. Further information about the gene, biosynthesis, release and receptors of MIPS are required to clarify the physiology of the muscle regulation by multiple neuromediators. Acknowledgements-We
wish to thank Dr T. Takao and Dr Y. Shimonishi for FAB-MS and Dr N. Iwasawa for peptide syntheses. We also thank Dr S. Sakata, M. Sakata, M. Furukawa, M. Yoshida, T. Kanda, N. Ohta, A. Miura, Y. Kuroki, K. Fujimoto, Y. Itoh, Y. Tanimura and M. Sugioka for collection and dissection of animals. A part of this work was supported by the Grant-in-Aid for General Scientific Research from the Ministry of Education, Science and Culture of Japan. REFERENCES
Fujisawa Y., Kubota I., Ikeda T. and Muneoka Y. (199Oa) Bioactive peptides isolated from the anterior byssus retractor muscle of the bivalve mollusc Myrilus edulis. In Peptide Chemistry 1989, (Edited by Yanaihara N.), pp. 51-56. Peptide Research Foundation, Osaka, Japan.
531
Fujisawa Y., Kubota I., Kanda T., Kuroki Y. and Muneoka Y. (199lb) Neuropeptides isolated from Myrilus edulis (Bivalvia) and Pusinur ferrugineus (Prosobranchia). In Comparative Aspects of Neuropeptide function. (Edited by Stefano G. B. and Florey E.), __ pp. 97-114. Manchester University Press, Manchester. Hirata T.. Kubota I.. Takabatake I.. Kawahara A.. Shimamoto N. and Muneoka Y. (1987) Catch-relaxing peptide isolated from Mytilus pedal ganglia. Brain Res. 422, 372-376.
Hirata T., Kubota I., Iwasawa N., Takabatake I., Ikeda T. and Muneoka Y. (1988) Structures and actions of Mytilus inhibitory peptides. Biochem. biophys. Res. Commun. 152, 13761382.
Hirata T., Kubota I., Iwasawa N., Fujisawa Y., Muneoka Y. and Kobayashi M. (1989) Effects of Mytilus inhibitory peptides on mechanical responses of various molluscan muscles. Comp. Biochem. Physiol. 93C, 381-388. Ikeda T.. Kiss T.. Hiriui L.. Fuiisawa Y.. Kubota I. and Muneoka Y. (199l)*MIP (M>tilus inhibitory peptide) analogues isolated from the ganglia of the pulmonate mollusc Helix pomatia. In Peptide Chemistry 1990 (Edited by Shimonishi Y.). pp. 357-362. Protein Research Foundation, Osaka, Japan. Kiss T. (1990) Effects of Mytilus inhibitory peptide on identified molluscan neurons. Comp. Biochem. Physiol. 95C, 207-2 12.
Kuroki Y., Kanda T., Kubota I., Fujisawa Y., Ikeda T., Miura A., Minamitake Y. and Muneoka Y. (1990) ~ , A molluscan neuropeptide related to the crustacean hormone, RPCH. Biochem. biophys. Res. Commun. 167, 273-279.
Muneoka Y. and Twarog B. M. (1977) Lanthanum block of contraction and of relaxation in response to serotonin and dopamine in molluscan catch muscle. J. Pharmac. exp. Ther. 202, 601609. Muneoka Y., Fujisawa Y., Fujimoto N. and Ikeda T. (1990) The regulation and pharmacology of muscles in Mytilus. In Neurobiology of Mytilus edulis (Edited by Stefano G. B.), pp. 209-245. Manchester University Press, Manchester. Muneoka Y., Fujisawa Y., Matsuura M. and Ikeda T. (1990) Neurotransmitters and neuromodulators controlling the anterior byssus retractor muscle of Mytilus edulis. Comp. Biochem. Physiol. C 98C, 105-l 14.
Camp. Biochem. Physiol.Vol. IOOC,No. 3, pp. 525-531, 1991
Printed in Great
Britain
A VARIETY THE ABRM
OF MYTKUS INHIBITORY PEPTIDES IN OF MYTKUS EDULIS: ISOLATION AND CHARACTERIZATION
YUKO FumAwA,*‘f ICHIRO KUBOTA,~ TETSUYAIEEDA, HIROYIJKI MINAKATA$ and YOJIRO MUNEOKA *Faculty of Integrated Arts and Sciences, Hiroshima University, Hiroshima 730, Japan; $Suntory Bio-Pharma Tech Center, Chiyoda-machi, Oura-gun, Gunma 370-05, Japan; and @untory Institute for Bioorganic Research, Shimamoto-cho, Mishimagun, Osaka 618, Japan (Received 21 December 1990)
Abstract-l. Five species of Mytilus inhibitory peptides, MIP,_,, were isolated from acetone extracts of the anterior byssus retractor muscle (ABRM) of Mytilus edulis. MIP, and MIP, were shown to be Sr-MIP and A2-MIP, respectively, first isolated from the pedal ganglia of the animal. 2. All the five peptides had a common C-terminal structure of -Pro-Xaa-Phe-Val-NH,, which was shown to be important for their biological activity. 3. The five MIPS showed similar inhibitory effects on contractions of the ABRM but did not affect catch tension and its relaxation. 4. In addition to the MIPS, catch-relaxing peptide (CARP) was also found in the ABRM.
INTRODUCTION In 1987 and 1988, three neuropeptides
MATERIALS AND were isolated
from the pedal ganglia of the bivalve mollusc Mytifus edulis (Hirata et al., 1987, 1988). One was termed “catch-relaxing peptide” (CARP) whose structure was determined to be Ala-Met-Pro-Met-Leu-ArgLeu-NH, . CARP showed a potent relaxing effect on catch tension of the anterior byssus retractor muscle (ABRM) of Mytilus. The other two were homologous peptides termed Mytilus inhibitory peptides (MIPS) after their remarkable inhibitory actions on contractions of the ABRM. Their structures were determined to be Gly-Ser-Pro-Met-Phe-Val-NH, (S*-MIP) and Gly-Ala-Pro-Met-Phe-Val-NH, (A*-MIP). Since the pedal ganglion is the main origin of the nerves innervating the ABRM (for review, see Muneoka et al., 1990), we expected that the peptides in the ganglion should be transported to, and stored in, the nerve terminals in the ABRM, to be released during neural excitation. Based on this expectation, we have attempted to isolate bioactive peptides from the ABRM. So far, we have found CARP, the two MIPS and also a number of other peptides including novel MIP-related peptides. At several conferences, we have presented their possible structures and representative actions (Fujisawa et al., 1990a, 1990b), but we have not yet published complete structure and detailed pharmacological and physiological characteristics of any of the peptides. Here we report the structures and actions of five species of MIPS and CARP isolated from the ABRM.
tTo whom correspondence should be addressed.
METHODS
Animals Mytilus edulis were collected from Hiroshima Bay and stored in a tank filled with aerated artificial seawater (ASW) at 17-24°C. For physiological experiments, animals were used within 7 days of collection, and for peptide purification, those within 10 hr were used. Meretrix lusoriu were purchased from a commercial source and stored in ASW. Euhadra congenita hiconis were collected at the campus of Hiroshima University. They were kept in the laboratory at 18-25°C and fed on sweet potatoes. Recordings of muscle activity
The isolated muscle preparation was suspended in a 1.5 ml polypropylene chamber. One end of the muscle was tied with a cotton thread and connected to a force transducer. The methods of stimulation and recording of contractions of the ABRM were essentially the same as those of Muneoka and Twarog (1977). The methods of recording of spontaneous contractions of the heart of Meretrix and the crop of Euhadra were essentially the same as those of Hirata et al. (1989) and Kuroki er al., respectively. Peptide purification
The ABRMs excised from 10,000 animals were homogenized in 100% and then 80% acetone (0.25 g tissue wet wtjml). The extracts were concentrated by a rotary evaporator, dissolved in 0.1 M HCI and forced through C,, cartridges (Waters, Sep-Pak). The retained material, eluted with 100% methanol, was applied to a Sephadex G-15 column (26 x 400mm) and eluted with 0.1 M acetic acid. Bioactivity of an aliquot from each fraction (4ml) was assayed on phasic contraction elicited by repetitive electrical pulses of stimulation (15 V, 3 msec, 10 Hz, for 5 set) and on catch tension produced by application of 10-“M acetylcholine (ACh) in the ABRM preparation. The gel-filtrated fractions which showed an activity on the ABRM were pooled and concentrated under reduced pressure. The concentrated material was injected into a C,, 525
YUKO
526
FUSAWA
Fig, 1. Anion-exchange HPLC profile of the second step of pu~~~~on of the ABRM extracts. Fractions indicated by A and B showed an inhibitory effect on phasic contraction of the ABRM. HPLC column (TSKgeI ODS-80T,, 4.6 x I50 mm, Tosoh) and eluted with a 60-min linear gradient of o-60% acetonitrile in 0.1% trifluornacetic acid (TFA) at pH 2.2. The active fractions from which the peptides reported here were purified were then injected into an con-exchange column frSKge1 DEAE-5PW, 7.5 x 75 mm, Tosoh) and elutedwith 10 mM T&ma-base at pH 9.6. The flowthrough fractions and the fractions eluted between 25 min and 35 min showed an inhibitory action on phasic contraction of the ABRM (Fig. 1). The tIow-through fractions (indicated by A in Fig. I) were injected into a cation-exchange cohmm (TSKgel SF-SPW, 7.5 x 75 mm, Tosoh) and eluted with a 58-min linear gradient of o-O.5 M NaCl in 20 mM phosphate buffer at pH 6.7. Several peaks of bioactivities were eluted at different positions. In the present experiments, further purification was carried out for peak I, II and III (Fig. 2). The fractions of each peak were separately applied to a C8 column (Finepak SIL CsS, 4.6 x 25Omm, Jasco) and eluted with a 40-min linear gradient of 2040% acetonitrile in 0.1% TFA. MIP, and MIPX were purified from peak I by two more steps of reversed-phase HPLC. CARP was purified from peak II by another step of reversed-phase HPLC. MIP, and MIP, were purified from peak III by one and two more steps of reversed-phase HPLC, respectively. The other group of inhibitory fractions obtained in the anion-exchange HPLC (indicated by B in Fig. I) was applied to the C, cc&mu and eluted with a 2&min linear gradient of 2025% acetonitrile in 0.1% TFA at pH 2.2 The inhibitory fractions were then applied to the C,, column and eluted isocratically with 20% acetonitrile in 0.1% TFA. MIPj was thus purified.
et al.
0
20
%Q
90 Tii
1~~
Fig. 2. ~a~on~x~h~~e HPLC profile of the next step of purification of fractions A in Fig, 1. Fractions I and III inhibited phasic contraction of the ABRM while fractions II potentiated it and relaxed catch tension caused by ACh. The HPLC charts of the &al purification for all the peptides are shown in Fig. 3.
All the purified substances underwent amino acid sequence analysis by the automated Edman degradation method with a gas-phase sequencer (Applied Biosystem 470A). Quantitative amino acid analysis was also performed for all the peptides. In addition, fast atom ~mbar~ent mass spectrometric (FAB-MS) measurement was carried out for MIP,, MIP, and MIFs, which had been supposed to be novel substances. Canjrmation of the proposed structures In order to confirm the structures proposed in the analyses, we compared the chemical and biological characteristics of each of the synthesized peptides to those of the native one. First, the behavior on the reversed-phase and the ion-exchange HFLC was compared. Secondly, the relation between doses and effects was compared in a ABRM preparation. Saline and drugs
The physiological saIine for the ABRM of ~~r~~~ and the heart of &ferer&x was ASW whose ~om~sition was: 445mM NaCl, IOmM KCl, 10mM Cat&, 55 mM MgCl, and 10 mM Tris-HCl (PH 7.8). The composition of the physiological saline far the land snail Euhadra was: 51 mM NaCl, 3.7mM KCl, 10mM CaCl,, 12.6mM MgClz, c
010
oTime
-
0
lo
(mini
Fig. 3. Reversed-phase HPLC profiles of the final step af purification, Arrows indicate u.v.-absorbant peaks of the peptides. a, MIP, ; b, MIP,; c, MIP,; d, MIP,; e, MIP,; f, CARP. The columns were TSKgeI ORS-SOT, (a-c) and Finepak SIL G5 (d-f). Each peptide was eluted isocratically with 0.1% TFA containing different concentrations of acetonitrile (a and b, 18%; c, 20%; d, 22%; e, 22%; f, 28%).
521
Mytilus inhibitory peptides in the ABRM 1.5 mM glucose and 10mM HEPESNaOH (PH 7.5). Acetylcholine bromide (ACh) was purchased from Sigma, and FMRFamidd, from Peninsula Labs Inc. CARP. MIP, (S2-MIP). MIP, (A2-MIP) and MIP fratzrnents (PMFVamidk‘and MFVamide) were synthesized byDr N. Iwasawa at the Suntory Institute for Biomedical Research (Osaka, Japan). Another MIP fragment, FVamide, was synthesized by Dr. H. Minakata at the Suntory Institute for Bioorganic Research. MIP,, MIP,, and MIP, were synthesized at the Peptide Institute (Osaka, Japan). Their structures were confirmed by the sequence analysis and the amino acid analysis. RESULTS
Structures of peptides The results of sequence analysis, amino acid analysis and FAB-MS were shown in Table 1. For MIP, , MIP, and MIP, , the C-terminal residue Val was not detected in the sequence analysis but the results of the amino acid analysis showed the presence of Val. The results of FAB-MS also suggested that these peptides had -Val-NH, at their C-terminal portions. Thus, we predicted the structures of the purified peptides as follows: MIP, (S2-MIP): Gly-Ser-Pro-Met-Phe-Val-NH, MIP, (A2-MIP): Gly-Ala-Pro-Met-Phe-Val-NH, MIP,: Asp-Ser-Pro-Leu-Phe-Val-NH, MIP,: Tyr-Ala-Pro-Arg-Phe-Val-NH, MIP,: Ala-Ser-His-Ile-Pro-Arg-Phe-Val-NH, CARP: Ala-Met-Pro-Met-Leu-Arg-Leu-NH, Each of the synthetic peptides with these structures showed the same profiles as those of the native one on the reversed-phase and the ion-exchange HPLC (Fig. 4). The bioactivity of the two peptides on the ABRM also coincided (Fig. 5). Thus, we concluded that the proposed structures are correct. The newly identified peptides, which were apparently homologous to S2-MIP and A*-MIP, were termed MIP,, MIP4 and MIP,, respectively, and S2-MIP and A2MIP were renamed MIP, and MIP,, respectively. It is notable that these five MIPS have in common Pro-Xaa-Phe-Val-NH, at their C-terminal parts.
Table 1. Results of structural Peptide MIP, MIP, MIP,
MIP,
MIP,
CARP
Effects of MIPS on the ABRM It has been shown that MIP, and MIP, inhibit contractions of the ABRM of Mytilus, e.g. phasic contraction caused by repetitive electrical pulses and tonic contractions caused by ACh and FMRFamide, and that the two peptides are almost equipotent in inhibiting these contractions (Hirata et al., 1988, 1989). In the present experiments, the newly isolated MIPS (MIP,, MIP, and MIP,) also inhibited the phasic contraction, ACh contraction and FMRFamide contraction (Fig. 6). The order of potency of the inhibitory effect on phasic contraction was MIP, > MIP, > MIP, > MIP,. In the case of ACh contraction, too, MIP, was more potent than MIP,, MIP, and MIP,, though MIP, was less effective than MIP, and MIP, In the case of FMRFamide contraction, the order of potency depended on preparations and significant difference in potency between the peptides was not observed. In some preparations, the FMRFamide contraction, after washing out MIPS, was more depressed than that recorded in the presence of the peptides (Fig. 6D). It has been also reported that MIP, and MIP, do not affect catchrelaxing response (Hirata et al., 1989). As well as these peptides, the three new MIPS neither relaxed catch tension nor changed relaxing response to repetitive electrical pulses of stimulation (Fig. 7). All the five members of MIP family conserve Pro-Xaa-Phe-Val-NH, at their C-terminal parts. We have shown that the C-terminal tetrapeptide (ProMet-Phe-Val-NH,) and even the tripeptide (Met-PheVal-NH,) of MIP, and MIP, can inhibit phasic contraction of the ABRM, although the effects are weaker than those of the native hexapeptides (Fujisawa et al., 1990b). In the present experiments, we confirmed these findings and also observed that even the dipeptide fragment, Phe-Val-NH,, which is common to all the five MIPS, showed an inhibitory effect on phasic contraction (Fig. 8). It is notable that the deletion of Pro residue from the tetrapeptide markedly reduced the inhibitory potency. Effects of MIPS on other molluscan muscles MIPS showed inhibitory effects not only on the ABRM but also on other molluscan muscles. They
analyses of the purified
peptides
Results of analyses + ) (I) Gly( 15O)-Ser(37)-Pro(35)-Met(4)-Phe(2)-Val( (2) Ser(l.03), GIy(1.07), Val(l.Ol), Met(O.‘ll), Phe(l), Pro(0.95) (I) Gly(l31)-Ala(lO3)-Pro(23)-Met(4)-Phe(3)-Val(+) (2) Gly(l.lS), Ala(1.09). Va1(1.04), Met(0.76). Phe(l), Pro(0.96) (1) Asp( I SO)-Ser(22)-Pro( 17)-Leu(S)-Phe(3) (2) Asp(1 .oO), Ser(0.91), Pro(0.93). Val(0.97), La(l), Phe(0.99) (3) 676.2 (676.4) (1) Tyr(l7)-Ala(21)-Pro(3l)-Arg(2)-Phe(9) (2) Ala(l.03). Vd(l), Tyr(0.92), Phe(0.96), Arg(1.33). Pro(0.96) (31 751.4 (751.5) ‘-’ &(146)-&( (I) 1 I)-His(4)-Ile(l9)-Pro(22)-Arg(7)-Phe(2) (2) Ser(0.94), Ala(0.96), Val(0.94), Ile(0.93), Phe(l), His(O.SS), Arg(l.04). (3) 925.3 (925.5) (1) Ala(444)-Met(262)-Pro(l27)-Met(l35)-Leu(l lO)-Arg(14)-Leu(33) (2) Ala(l.OE), Met(1.37), Leu(2). Arg(1.25), Pro(+)
Pro(0.78)
(1) Amino acid sequence analysis. Each value means yield of PTH-amino acids (pmol). (2) Amino acid analysis. Each value means relative amount of amino acids normalized on the underlined amino acid indicated by italic. (3) FAB-MS measurement. The values are m/z of molecular ion peaks, indicating molecular weight plus one for H+. Theoretical value for each peptide (amidated form) was shown in ( ).
528
YUKO FUJISAWA et al.
.008
r
1
CARP
Fig. 4. Comparison of retention time on reversed-phase HPLC between native and synthesized peptides. Each of the native peptides (N, upper trace) and the synthetic peptide (S, middle trace) were eluted at the same retention time. The mixture of the two was co-eluted as a single u.v.-absorbant peak (N + S, lower trace). Similar results were obtained on ion-exchange HPLC (not shown). loo-
100
MlP*
. . / ./ .
2 gso._
:/
iG = 5
OL
8
3x1o’8
3x10-9
10-4 Concentration (MI
/
I
o-,
1
10-Q
MIPJ 1
3x10’0
10-P
I 3x10-4
concentmtii
10-a
(MI
oL, 3xlo-s
1
lo-a
#
3x10-8
Concentratiin (M)
50MIP,
I
6
MIPS
-1 d .
7 /
s 3 30 g
25/
B
./’ . OL, 3x10.9
I
10-a
I
I
3x10-4
concentmtii
(M)
10”
/
0
<,
10-s
,(
3x10-’ 3x10-s 10-7 Concentmtion (M)
Fig. 5. Comparison of bioactivities on the ABRM between native and synthesized peptides. The inhibitory effects of MIPS were examined on phasic contraction. Decrease in peak tension was expressed by per cent of the control peak tension. The relaxing activity of CARP was examined on catch tension caused by 10e4 M ACh. Decrease in catch tension during exposure to CARP for 4 min was expressed by percent of the tension just before applying CARP. 0, Native peptides; a, synthesized peptides.
I
10”
Mytilus inhibitory peptides in the ABRM
529
Fig. 6. Inhibitory effects of MIPS on the ABRM. A: effects on phasic contraction elicited by repetitive electrical pulses. The electrical stimulation was applied every 10 min. MIPS were applied 8 min prior to the contractions (upward arrows) and washed out soon after them (downward arrows). B: effects on ACh contraction. ACh 10e5 M was applied for 2 min every 20 min. MIPS were applied 10 min prior to the ACh contractions (upward arrows) and washed out soon after them (downward arrows). After each recording, catch tension was relaxed by repetitive electrical pulses. C: effects on 10e6 M FMRFamide contraction. Experimental procedure was similar to that in B. D: an example of the “outlasted inhibitory effect” of MIPS. Note that the peak tension after washing out MIP is smaller than that during exposure to the peptide.
inhibited the cardiac activity of the bivalve Meretrix Iusoria (Fig. 9A) and the spontaneous activity of the crop of the pulmonate Euhadra congenita hiconis (Fig. 9B). In the heart of Meterix, inhibitory effects of MIPS considerably varied with preparations. In each of the animals, the five peptides showed qualitatively similar inhibitory effects, though they were not equipotent. MIP, was the most potent both in Meretrix and in Euhadra; it markedly and in some
Fig. 7. Effects of MIPS on relaxation of the ABRM by repetitive electrical pulses (15 V, 3 msec, 1 Hz, for 10 set). MIPS did not cause relaxation of catch by themselves and did not affect catch-relaxing response to electrical stimulation (ES).
preparations completely depressed the spontaneous activities of the heart and the crop at low doses such as lo-* to lo-‘M. DISCUSSION
The ABRM of Mytilus was shown to contain CARP and five species of MIPS. These peptides are supposed to be present in the nerve elements in the ABRM and to act as physiological neuromediators. Especially, the presence of CARP, MIP, and MIP,, both in the pedal ganglion and in the ABRM, strongly supports the notion that they are the neuropeptides synthesized in the ganglionic cell body, transported to the nerve terminals and released to the muscle fibers during neural excitation. The origin of the other three MIPS have not been clarified yet but it is possible that they are also contained in the pedal ganglion. In order to establish physiological roles of these peptides, further experiments including electrophysiological and histochemical ones are required. All the five MIPS isolated from the ABRM are closely related to each other both in their structure and biological activity. They exhibit qualitatively similar effects on the ABRM; that is, they exclusively inhibit the contractions and do not affect the
530
YUKOFUJISAWA et al.
~&~~~~/Q 5
PYFVa
MIPZ
MFVa
MFVa
M
:“;;nm
FVa
Fig. 8. Inhibitory effectsof C-terminal fragments of MIPs on phasic contraction of the ABRM in response to repetitive electrical pulses of stimulation. Note that the deletion of Pro from PMFVamide markedly decreases the inhibitory potency. relaxation of catch. The structure common to the five peptides was found to be important for these inhibitory effects; the C-terminal dipeptide Phe-Val-NH, is thought to be essential, and another common residue Pro, which is situated at the forth position from the C terminus, seems to play an important role. These findings may suggest that the five MIPS interact with the same class of receptors which requires the tetrapeptide sequence. Pro-Xaa-Phe-Val-NH,. The other residues, which seem not to be crucial to receptor-ligand interaction, may cause the quantitative difference in inhibitory potency of the five MIPS; they affect the ability of peptide molecules to bind the receptors and the resistance to degradation enzymes. Thus, it can be supposed that the structural variety in MIPS have little physiological importance and that the five analogous MIPS are recognized as the same kind of molecules within the MIP-operating system. If this is true, the MIP system could offer an hypothesis concerning the evolution of signal molecules; when a variation was produced from an ancestral form by gene duplication and mutation, it could be conserved unless the change in the molecule severely affects the ability to bind its receptors. Whether this is true or not, a great deal of information are required A. Meretrix
to answer the question why such many analogs are involved in the regulation of a muscle. It has been proposed that MIP systems are distributed in other molluscs since MIP, and/or MIP, have inhibitory effects on the heart beat of the bivalve, Tapes japonica, twitch and tetanic contractions of the penis retractor muscle of the pulmonate, Achatinafulica (Hirata er al., 1988, 1989) and also on the identified neurones of Achatina (Yongsiri et al., 1989) and of Helix pomatia (Kiss, 1990). In the present experiments, the five members of MIPS showed similar effects on each of Meterix and Euhadra, suggesting that these animals may also have a class of receptors with which a variety of MIP-like peptides interacts, as in the case of Mytilus. Recently, Ikeda et al. isolated as many as 13 MIP analogs from the central nervous system of Helix by using the ABRM as a bioassay system (Ikeda et al., 1991). They have -Pro-Xaa-Phe-Val-NH, except one peptide with -Pro-Xaa-Phe-Ile-NH,. In a prosobranch mollusc Fusinus ferrugineus, however, MIPS of Mytilus showed little or no effect on contractions of the radula retractor muscle and the proboscis retractor muscle (unpublished). In addition, we have failed to isolate MIP-like peptides from the acetone extracts of
heart
MIP3
IO-’ M MIP, 0.59 lmin
MlPl 0. Euhadra crop
IO-’ M MIPI
MlP2
1
MIP3
MIPS Fig. 9. Effects of MIPS on other molluscan muscles. A: effects on the heart beat of Meretrix. B: effects on the spontaneous activity of the crop of Euhadra.
Mytilus inhibitory peptides in the ABRM the central ganglion masses of Fusinus by using the radula muscle as a bioassay system. From a view point of phylogeny, however, prosobranchia and opisthobranchia can be expected to have MIP-related peptides as well as bivalvia and pulmonata. Study on the distribution of MIP-related peptides in other classes of molluscs should be done by using the ABRM as a bioassay system. In addition to CARP and the five MIPS, we have found many other bioactive peptides including at least two MIP-like peptides (unpublished) in the ABRM, though their structures have not been completely confirmed yet (Fujisawa et al., 1990a,b). The ABRM of Mytilus might be a good example of the muscles which are regulated by a number of neuropeptides as well as by the classical neurotransmitters such as ACh or S-HT. Involvement of multiple analogous peptides which seem to exert the same kind of action might be one of the interesting phenomena in such a muscle. Further information about the gene, biosynthesis, release and receptors of MIPS are required to clarify the physiology of the muscle regulation by multiple neuromediators. Acknowledgements-We
wish to thank Dr T. Takao and Dr Y. Shimonishi for FAB-MS and Dr N. Iwasawa for peptide syntheses. We also thank Dr S. Sakata, M. Sakata, M. Furukawa, M. Yoshida, T. Kanda, N. Ohta, A. Miura, Y. Kuroki, K. Fujimoto, Y. Itoh, Y. Tanimura and M. Sugioka for collection and dissection of animals. A part of this work was supported by the Grant-in-Aid for General Scientific Research from the Ministry of Education, Science and Culture of Japan. REFERENCES
Fujisawa Y., Kubota I., Ikeda T. and Muneoka Y. (199Oa) Bioactive peptides isolated from the anterior byssus retractor muscle of the bivalve mollusc Myrilus edulis. In Peptide Chemistry 1989, (Edited by Yanaihara N.), pp. 51-56. Peptide Research Foundation, Osaka, Japan.
531
Fujisawa Y., Kubota I., Kanda T., Kuroki Y. and Muneoka Y. (199lb) Neuropeptides isolated from Myrilus edulis (Bivalvia) and Pusinur ferrugineus (Prosobranchia). In Comparative Aspects of Neuropeptide function. (Edited by Stefano G. B. and Florey E.), __ pp. 97-114. Manchester University Press, Manchester. Hirata T.. Kubota I.. Takabatake I.. Kawahara A.. Shimamoto N. and Muneoka Y. (1987) Catch-relaxing peptide isolated from Mytilus pedal ganglia. Brain Res. 422, 372-376.
Hirata T., Kubota I., Iwasawa N., Takabatake I., Ikeda T. and Muneoka Y. (1988) Structures and actions of Mytilus inhibitory peptides. Biochem. biophys. Res. Commun. 152, 13761382.
Hirata T., Kubota I., Iwasawa N., Fujisawa Y., Muneoka Y. and Kobayashi M. (1989) Effects of Mytilus inhibitory peptides on mechanical responses of various molluscan muscles. Comp. Biochem. Physiol. 93C, 381-388. Ikeda T.. Kiss T.. Hiriui L.. Fuiisawa Y.. Kubota I. and Muneoka Y. (199l)*MIP (M>tilus inhibitory peptide) analogues isolated from the ganglia of the pulmonate mollusc Helix pomatia. In Peptide Chemistry 1990 (Edited by Shimonishi Y.). pp. 357-362. Protein Research Foundation, Osaka, Japan. Kiss T. (1990) Effects of Mytilus inhibitory peptide on identified molluscan neurons. Comp. Biochem. Physiol. 95C, 207-2 12.
Kuroki Y., Kanda T., Kubota I., Fujisawa Y., Ikeda T., Miura A., Minamitake Y. and Muneoka Y. (1990) ~ , A molluscan neuropeptide related to the crustacean hormone, RPCH. Biochem. biophys. Res. Commun. 167, 273-279.
Muneoka Y. and Twarog B. M. (1977) Lanthanum block of contraction and of relaxation in response to serotonin and dopamine in molluscan catch muscle. J. Pharmac. exp. Ther. 202, 601609. Muneoka Y., Fujisawa Y., Fujimoto N. and Ikeda T. (1990) The regulation and pharmacology of muscles in Mytilus. In Neurobiology of Mytilus edulis (Edited by Stefano G. B.), pp. 209-245. Manchester University Press, Manchester. Muneoka Y., Fujisawa Y., Matsuura M. and Ikeda T. (1990) Neurotransmitters and neuromodulators controlling the anterior byssus retractor muscle of Mytilus edulis. Comp. Biochem. Physiol. C 98C, 105-l 14.