Mytilus Inhibitory Peptides Research Articles

Peptidergic innervation of the vasoconstrictor muscle of the abdominal aorta inAplysia kurodai

The arterial system of the marine mollusc Aplysia consists of three major arteries. One of them, the abdominal aorta, has a sphincter (the vasoconstrictor muscle) at the base of the artery. Contraction of this muscle reduces the blood flow into the abdominal aorta, thereby, playing a role in the regulation of the blood distribution in Aplysia. Here, we show the contractility of the vasoconstrictor muscle is modulated by three types of endogenous peptides, Aplysia mytilus inhibitory peptide-related peptides (AMRP), enterin and NdWFamide. Immunohistochemistry showed that putative neuronal processes containing the three peptides exist in the vasoconstrictor muscle. Enterin inhibited the muscle contraction elicited by the nerve stimulation or the application of a putative excitatory transmitter, acetylcholine (ACh). Enterin hyperpolarized the resting potential of the muscle and decreased the amplitude of the excitatory junction potential (EJP). AMRP also inhibited the nerve-evoked contraction although its action on the ACh-induced contraction was variable. AMRP also reduced the size of EJP, but had no effect on the resting potential of the muscle. NdWFamide enhanced the nerve-evoked contraction but not the ACh-induced contraction. NdWFamide augmented EJP without affecting the resting potential of the muscle. These results suggest that AMRP, enterin and NdWFamide are endogenous modulators of the contractile activity of the vasoconstrictor muscle, and that the peptidergic innervations of this muscle contribute to fine tuning of the blood distribution in Aplysia.

Structural and functional diversities of the Aplysia Mytilus inhibitory peptide-related peptides

Aplysia Mytilus inhibitory peptide-related peptides (AMRPs) are multiple hexapeptides coded on a single precursor. By comparing the AMRP precursors of two species of Aplysia ( Aplysia californica and Aplysia kurodai), we found that there are substantial numbers of species-specific AMRPs. We next compared the function of AMRPs on the anterior aorta between A. kurodai and Aplysia juliana. In A. juliana, AMRPs inhibited the contractile activity of the aorta (EC 50=10 −9 to 10 −8 M), whereas the peptides had no obvious action in A. kurodai up to 10 −7 M. These results indicate that AMRPs are both structurally and functionally diverse neuropeptides even among closely related species.

Mytilus inhibitory peptides (MIP) in the central and peripheral nervous system of the pulmonate gastropods, Lymnaea stagnalis and Helix pomatia: distribution and physiological actions.

The distribution and neuroanatomy of Mytilus inhibitory peptides (MIP)-containing neurons in the central nervous system and their innervation pattern in the peripheral nervous system of the pulmonate snail species, Lymnaea stagnalis and Helix pomatia, have been investigated immunocytochemically, by applying an antibody raised to GSPMFVamide. A significant number of immunoreactive neurons occurs in the central nervous system of both species (Lymnaea: ca 600-700, Helix: ca 400-500), but their distribution is different. In Lymnaea, labeled neurons are found in all central ganglia where a number of large and giant neurons, previously identified physiologically, reveal MIP immunoreactivity. In Helix, most of the immunolabeled neurons are small (12-30 microm) and concentrated in the buccal and cerebral ganglia; the parietal ganglia are free of labeled cells. In both species, the ganglionic neuropils, peripheral nerves, connectives, and commissures are richly supplied with immunolabeled fibers. The MIP-immunoreactive innervation pattern in the heart, intestine, buccal mass and radula, and foot is similar in both species, with labeled axonal bundles and terminal-like arborizations (buccal mass, foot) or a network of varicose fibers (heart, intestine). Intrinsic neurons are not present in these tissues. The application of GSPYFVamide inhibits the spontaneous contractions of the esophageal longitudinal musculature in Helix, indicating the bioactivity of the peptide. An outside-out patch-clamp technique has demonstrated that GSPYFVamide opens the K+ channels in central nerve cells of Helix. Injection of GSPYFVamide into the body cavity inhibits the feeding of starved Helix. A wide modulatory role of MIP at central and peripheral levels is suggested in Lymnaea and Helix, including the participation in intercellular signalling processes and remote neurohormonal-like control effects.

Ionic mechanism mediating Mytilus inhibitory peptides elicited membrane currents in identified Helix neurons

Effects of seven, pressure applied MIP ( Mytilus inhibitory peptides) had been studied on D-neurons of the CNS of Helix pomatia in voltage-clamp experiments. In physiological saline, the peptides produced a hyperpolarization usually coupled with the cessation of any spontaneous spiking activity. Clamped at the resting potential (∼−60 mV), peptide applications elicited an outward current, which increased its amplitude by shifting the holding potential towards depolarisation. The response was concentration-dependent and accompanied by an increased membrane conductance. Reversal potentials obtained at different [K +] o were plotted with a slope of 52 mV per ten-fold change in [K +] o showing that the peptide-elicited current was mainly due to the increased K +-conductance(s). The peptide-induced outward current could partially be blocked by Ba 2+ (5 mM), CdCl 2 (1 mM), TEACl (10 mM) or apamin (2.5×10 −5 M) or furosemide (10 mg/ml) and decreased either in Na +-free or Cl −-free solutions. 4-Aminopyridine at 5 mM concentration completely blocked the peptide-induced current. In the presence of high [K +] o, the peptide(s) was still found to induce an outward current at membrane potentials beyond K +-reversal potential. This component was not present in Cl −-free saline, suggesting that the current was due to the inward flow of Cl − ions. Our results show that the MIPs have at least two (three) independent actions, each associated with different voltage-, concentration-dependence and ionic mechanisms. It is suggested, that the peptide-induced currents are carried by K +, and Cl − ions. According to our present finding, the observed effects are mediated by the same receptor, activating different second messenger systems, inducing multiple conductance changes in the membrane of neurons of the snail ganglia.

Structural requirements and ionic mechanism of the mytilus inhibitory peptides (MIPs) on Helix central neurons

1. 1. The structural and ionic requirements for potent FVamide-induced inhibition were investigated using Helix aspersa central neurons under current or voltage clamp. 2. 2. For potent FVamide inhibition the full hexapeptide sequence, GSPYFVamide, was required. The rank order of decreasing potency was GSPYFVamide>>SPYFVamide>PYFVamide>YFVamide. 3. 3. GSPYFVamide inhibition involved an increase in conductance to both potassium and chloride ions as demonstrated by ion substitution experiments and addition of 4-aminopyridine, tetraethylammonium, 4,4-di-isothiocyanatostilbene-2,2'-disulfonic acid, 4,4-dinitrostilbene-2,2'-disulfonic acid disodium salt and furosemide to the solution bathing the preparation.

Immunohistochemical Localization and Ca2+-Dependent Release ofMytilusInhibitory Peptides in the ABRM of Mytilus edulis

Abstract Immunohistochemical localization of Mytilus inhibitory peptides (MIPs) in the anterior byssus retractor muscle (ABRM) of Mytilus edulis was investigated by using the anti-MIP polyclonal antibody. The antibody was shown to recognize the seven members of the MIP family that had been previously isolated from the ABRM extract. The MlP-like immunoreactivity was found abundantly in neuronal fiber-like structures in the ABRM and in the connective tissue sheath covering it. The immunoreactive fibers in both areas were rich in varicosities. In addition, it was demonstrated that the MlP-like immunoreactivity was released from the neuromuscular preparation to the bathing solution in response to the repetitive electrical pulses applied to the pedal ganglion. The release was Ca2+ dependent. These findings suggest that the seven MlP-family peptides, originally isolated from the muscle extract, are the inhibitory neuropeptides involved in physiological regulation of the ABRM contraction.

A Novel cDNA Sequence Encoding the Precursor of the d‐Amino Acid‐Containing Neuropeptide Fulicin and Multiple α‐Amidated Neuropeptides from Achatina fulica

Fulicin (Phe-D-Asn-Glu-Phe-Val-NH2) is an endogenous neuropeptide containing a D-amino acid from ganglia of the African giant snail Achatina fulica. We have cloned a novel cDNA (1,995 nucleotides) encoding a fulicin precursor from the snail cerebral and subesophageal ganglia. The fulicin precursor protein (357 amino acids) contains one copy of fulicin and at least nine other putative alpha-amidated neuropeptides composed of four to six amino acid residues. Seven of the nine neuropeptides were novel, and the other two had the same structures as Mytilus inhibitory peptide-related peptides previously isolated from the ganglia of Helix pomatia. All sequences of 10 peptides are flanked by Lys-Arg(Lys) at the N-terminus and by Gly-Lys-Arg(Lys) at the C-terminus. Nucleotide sequence analysis revealed that D-Asn present in fulicin is encoded by the usual L-Asn codon (AAT). Although fulicin has as yet only been isolated from the central ganglia. RNA blot analysis revealed that single transcripts of approximately 2.0 kb in size also exist in the ventricles and atria. These results suggest that fulicin and related peptides are produced in neurons and the heart by the processing of a ribosomally made precursor, although the mechanism of in-chain epimerization remains unclear.

Mytilus-inhibitory peptide analogues isolated from the ganglia of a pulmonate mollusc, Achatina fulica

1. Ten peptides that showed an inhibitory effect on phasic contraction of the ABRM of Mytilus were isolated from the ganglia of the African giant snail, Achatina fulica. 2. Seven of the peptides were shown to be hexapeptides having -Pro-Xaa-Phe-Val-NH 2 as a common structure, which was previously shown to be a characteristic of Mytilus inhibitory peptides (MIPs). 3. The remaining three were pentadecapeptides, each of which consisted of two MIP-related hexapeptides linked by -Gly-Arg-Arg-.

Neurotransmitters and neuromodulators controlling the anterior byssus retractor muscle of Mytilus edulis

1. The anterior byssus retractor muscle (ABRM) of Mytilus edulis is innervated by at least two kinds of nerves, excitatory and relaxing nerves. The principal neurotransmitters released from these nerves have been shown to be acetylcholine and serotonin, respectively. 2. Some other monoamines, such as dopamine and octopamine, and various peptides, such as FMRFamide-related peptides, Mytilus inhibitory peptides, SCP-related peptides and a catch-relaxing peptide, may also be involved in the regulation of the muscle as neurotransmitters or neuromodulators. 3. The ABRM seems to be typlcal of invertebrate muscles controlled by multiple neurotransmitters and neuromodulators.

A variety of Mytilus inhibitory peptides in the abrm of Mytilus edulis: Isolation and characterization

1. Five species of Mytilus inhibitory peptides, MIP 1–5, were isolated from acetone extracts of the anterior byssus retractor muscle (ABRM) of Mytilus edulis. MIP 1 and MIP 2 were shown to be S 2-MIP and A 2-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 2, 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.

Effects of Mytilus inhibitory peptides on a giant neurone of Achatina fulica Férussac

Among several identifiable neurones of Achatina fulica Férussac, RAPN (right anterior pallial neurone) was sensitive to the two Mytilus inhibitory peptides (MIPs), H-Gly-Ser-Pro-Met-Phe-Val-NH 2 ([Ser 2]MIP) and H-Gly-Ala-Pro-Met-Phe-Val-NH 2 ([Ala 2]MIP), and their fragments, H-Pro-Met-Phe-Val-NH 2 (MIP-(3–6)) and H-Met-Phe-Val-NH 2 (MIP-(4–6)). These peptides, applied either locally by pneumatic pressure or in the bath, produced a slow outward current with an increase in membrane conductance. The other two related peptides, H-Gly-Ala-Pro-Met-Val-Phe-NH 2 ([Ala 2, Val 5,Phe 6]MIP-NH 2) and H-Gly-Ala-Pro-Met-NH 2 ([Ala 2]MIP-(1–4)-NH 2), were ineffective. The potency order of the four effective peptides was determined from their dose-response relations: [Ser 2]MIP > [Ala 2]MIP > MIP-(3–6) > MIP-(4–6). The ED 50 of [Ser 2]MIP was about 3 × 10 −5 M. Relations between the outward current (nA) and the conductance increase (μS) produced by the four peptides were quite linear (Y = 0.03416 × −0.01083, r = 0.98677). The reversal potentials for [Ser 2]MIP (E S-MIP) measured with various extracellular K + concentrations ([K +] 0) were fitted to the Nernst equation, being identical with E K. E S-MIP was not affected by changing [Na +] 0 and [Cl −] 0.

Effects of Mytilus inhibitory peptides on mechanical responses of various molluscan muscles

1. Effects of the Mytilus inhibitory peptides (MIPs), Ala 2-MIP and Ser 2-MIP, on mechanical responses of various molluscan muscles were examined. 2. In the ABRM of Mytilus edulis, all contractions in response to repetitive electrical pulses of stimulation, ACh, FMRFamide and caffeine were inhibited by MIPs. Contraction by 400 mM K + ASW was invariably not affected, but that by 100 mM K + ASW was slightly inhibited in some muscles. 3. In the pedal retractor muscle of Mytilus edulis, contractions in response to repetitive electrical pulses of stimulation and ACh were also inhibited by MIPs. 4. In the heart of Meretrix lusoria, its activity was inhibited by MIPs. Ala 2-MIP was found to be more potent than Ser 2-MIP. The ACh-antagonist benzoquinonium did not change the inhibitory effects of the peptides. In the heart of Tapes japonica, both of the peptides showed weak inhibitory effect at 100 nM. At 10 μM, however, Ala 2-MIP showed inhibitory effect followed by excitatory effect, and Ser 2-MIP showed excitatory effect and then brought about a transient cystolic arrest. 5. Twitch and tetanic contractions of the penis retractor muscle of Achatina fulica were inhibited by MIPs. Ala 2-MIP was found to be more potent than Ser 2-MIP. 6. MIPs seem to have an inhibitory effect on many molluscan muscles. There may exist a family of MIP-related peptides in molluscs.

Structures and actions of Mytilus inhibitory peptides

Two congeneric peptides that inhibit contraction of the anterior byssus retractor muscle of Mytilus edulis were isolated from the pedal ganglia of the mussel. Their structures were determined to be H-Gly-Ser-Pro-Met-Phe-Val-NH2 and H-Gly-Ala-Pro-Met-Phe-Val-NH2. These hexapeptides also showed inhibitory action on contractions in several other molluscan muscles, such as the cardiac muscle of Meretrix lusoria and the penis retractor muscle of Achatina fulica.