Further study of effects of synthetic peptides on identifiable giant neurones of an African giant snail (Achatina fulica Férussac)

1. Effects of the following peptides at 10(-4) M on identifiable giant neurones of Achatina fulica Férussac were examined: physalaemin, eledoisin, bradykinin, neurokinin A, neurokinin B, neuromedin B, gastrin releasing peptide decapeptide (neuromedin C), gastrin releasing peptide (14-27), cholecystokinin tetrapeptide, cholecystokinin octapeptide, thyrotropin releasing hormone, Arg-vasotocin, gamma-melanocyte stimulating hormone. 2. The six neurones tested were as follows: PON (periodically oscillating neurone), TAN (tonically autoactive neurone), RAPN (right anterior pallial neurone), d-RPLN (dorsal-right parietal large neurone), VIN (visceral intermittently firing neurone) and d-VLN (dorsal-visceral large neurone). 3. Of the peptides examined, only Arg-vasotocin at 10(-4) M produced the excitatory effects on PON, VIN and d-VLN. Physalaemin showed slight inhibitory effects on TAN; this substance was sometimes almost ineffective on the neurone. 4. The other peptides examined were completely ineffective on all of the neurones tested.     

Further study of effects of synthetic peptides on identifiable giant neurones of an african giant snail (Achatina fulica Férussac)

• 1.1. Effects of the following peptides at 10⁻⁴ M on identifiable giant neurones of Achatina fulica Férussac were examined: physalaemin, eledoisin, bradykinin, neurokinin A, neurokinin B, neuromedin B, gastrin releasing peptide decapeptide (neuromedin C), gastrin releasing peptide (14–27), cholecystokinin tetrapeptide, cholecystokinin octapeptide, thyrotropin releasing hormone, Arg-vasotocin, γ-melanocyte stimulating hormone.
• 2.2. The six neurones tested were as follows: PON (periodically oscillating neurone), TAN (tonically autoactive neurone), RAPN (right anterior pallial neurone), d-RPLN (dorsal-right parietal large neurone), VIN (visceral intermittently firing neurone) and d-VLN (dorsal-visceral large neurone).
• 3.3. Of the peptides examined, only Arg-vasotocin at 10⁻⁴ M produced the excitatory effects on PON, VIN and d-VLN. Physalaemin showed slight inhibitory effects on TAN; this substance was sometimes almost ineffective on the neurone.
• 4.4. The other peptides examined were completely ineffective on all of the neurones tested.

     

Inhibitory effects of dipeptides containing tryptophan on the excitability on the giant neuron of the giant African snail (Achatina fulica, Ferussac)]

Besides an inhibitory effect of L-Lys-L-Phe-L-Tyr and L-Phe-L-Tyr, L-Phe-L-Trp showed the same effect on the excitability of a giant neurone (the TAN, tonically autoactive neurone) identified in suboesophageal ganglia of Achatina futica Férussac. The critical concentration of L-Phe-L-Trp to produce the effect was 10(-5)--3 X 10(-5) kg/1. The inhibitory effect of this substance was relatively slow and independent no chloride ions, as well as in the case of L-Phe-L-Tyr. L-Trp-L-Phe, Gly-L-Trp and L-Trp-Gly did not show any effect on the same neurone. The I-V curve of the TAN's neuromembrane, measured by the injection of a transmembrane triangular current, were not modified markedly by L-Phe-L-Trp at 4 X 10(-4) kg/1. The pattern of the TAN's spike firing produced by the depolarizing current injection were not influenced by this substance.     

Isolation of achatin-I, a neuroactive tetrapeptide having a D-phenylalanine residue, from Achatina ganglia, and its effects on Achatina giant neurones.

1. We have isolated a neuroexcitatory tetrapeptide having a D-phenylalanine (Gly-D-Phe-L-Ala-L-Asp) from the ganglia of Achatina fulica Férussac. This peptide was termed achatin-I (Kamatani et al., 1989). In the present report, we shall present highlights from the original paper concerning the process of peptide isolation and the examination of its effects. 2. From the ganglia of about 30,000 animals, we obtained 50 micrograms of achatin-I and 17 micrograms of its stereoisomer consisting of only L-amino acid residues (Gly-L-Phe-L-Ala-L-Asp) which was termed achatin-II. The data of instrumental analyses (1H-NMR, SIMS, CD and HPLC) of isolated achatin-I and achatin-II were identical to those of synthetic ones. 3. Achatin-I showed marked excitatory effects on the three Achatina giant neurones, PON (periodically oscillating neurone), TAN (tonically autoactive neurone) and v-RCDN (ventral-right cerebral distinct neurone), whereas achatin-II had no effect. Among their stereoisomers, [D-Ala3]-achatin-I (Gly-D-Phe-D-Ala-L-Asp) had slight excitatory effects on the Achatina neurones tested. Amide derivatives of achatin-I and achatin-II were ineffective. 4. Dose-response curves of achatin-I and [D-Ala3]-achatin-I for producing the inward current of PON were measured under voltage clamp at a holding membrane voltage (Vh) of -50 mV.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of clavines, ergot alkaloids, on the membrane potential of an identifiable giant neuron of the African giant snail Achatina fulica Ferussac

The effects of seven clavines, alkaloids of ergot, on the electrical activity of an identifiable giant neurone (TAN, tonically autoactive neurone) of the African giant snail were examined. All the substances examined, lysergine, agroclavine, elymoclavine, festuclavine, chanoclavine, rugulovasine A and rugulovasine B, at 2 X 10(-4) kg/l have no constant effect on TAN, indicating that they have no direct effect on this neurone. However, the substances examined, except for chanoclavine, in the same concentration occasionally caused the transient depression with an augmentation of trans-synaptic influences. This depression may be due to the trans-synaptic influences. The four substances examined, lysergine, agroclavine, elymoclavine and festuclavine, in the same concentration produced TAN abnormal spike discharges, doublet or triplet spikes.     

The characteristics of the cholinoreceptors on the identified TAN neuron of the giant African snail Achatina fulica]

Acetylcholine, nicotine, a selective agonist of N-cholinoreceptors suberildicholine dibromide, as well as a selective agonist of M-cholinoreceptors 5-methylfurmethide inhibited spike discharges in a dose-dependent manner up to a complete ceasing of the firing in cholinoreceptors situated on the identified neurone TAN of African giant snail Achatina fulica. M-cholinoblocker metamizylum completely prevented the inhibitory effect of methylfurmethide. Central cholinoblocker aetherophen completely prevented the inhibitory effect of suberildicholine dibromide. Metamizylum or aetherophen used alone were only able to decrease the inhibitory effect of acetylcholine, whereas a mixture of these agents suppressed completely the acetylcholine-induced inhibition. The findings suggest that, on the TAN membrane, nicotinic and muscarinic cholinoreceptors co-exist and function in one and the same direction.     

The effect of amino acids on the excitability of identified autoactive giant neurons of Achatina fulica Férussac]

Effects of the following amino acids were examined on the electrical activity of the two giant neurones (PON and TAN) identified in the subesophageal ganglia of Achatina fulica Férussac : L-Asp, L-Thr, L-Ser, L-Glu, L-Pro, Gly, L-alpha-Ala, beta-Ala, L-cysteine, L-cystine, L-Val, L-Met, L-Ileu, L-Leu, L-Tyr, L-Phe, L-Lys, L-His, L-Arg, L-Cit, L-Try, GABA and GABOB. Among these substances, we observed an inhibitory effect of GABA and GABOB on the TAN excitability. GABA showed stronger effect on the TAN than GABOB. This effect of GABA was due to producing hyperpolarization on the TAN membrane. GABA showed a slight excitatory effect on the PON. The effect of GABOB on the PON was very weak and unstable.     

Effects of synthetic ergot derivatives on the two identifiable giant neurons, sensitive to dopamine, of Achatina fulica Ferussac

On the two dopamine (DA)-sensitive giant neurones, PON (periodically oscillating neuron, excited by DA) and TAN (tonically autoactive neuron, inhibited by DA), of an African giant snail (Achatina fulica Férussac), effects of synthetic ergot derivatives, including lisuride and pergolide, which are considered to be dopamine agonists, were examined. Of the substances examined, three of the ergot derivatives related to pergolide, D-8,9-didehydro-6-propylergoline-8-methanol (LY149174), D-6-methyl-8 beta-(2-(methylsulfinyl)ethyl)ergoline (LY116470) and D-2-chloro-6-methyl-8 beta-(2-(methylsulfinyl)ethyl)ergoline (LY127817), showed excitatory effects on PON, while pergolide (D-8 beta-( (methylthio)methyl)-6-propylergoline, LY127809) and lisuride (N-D-6-methyl-8-isoergolenyl-N',N'-diethylcarbamide) had no effect. On the other hand, only D-6-methyl-8 beta-(2-(methylsulfinyl)ethyl)ergoline (LY116470) had any excitatory effects on TAN.     

Effects of histamine and its derivatives on the excitability of a giant identifiable neuron of Achatina fulica Férussac. A histaminergic receptor different from H1 and H2]

A giant neurone of Achatina fulica Férussac (the TAN, tonically autoactive neurone) is excited by histamine. Pharmacological characteristics of its histaminergic reception are quite different from those of H1 and H2 receptors. The effect of histamine on the TAN is antagonized by neither mepyramine nor burimamide.     

Effects of 2 water-soluble contrast media, meglumine iothalamate (Conray) and meglumine iocarmate (Dimer-X), on the electric activity of identifiable giant neurons of the African giant snail (Achatina fulica Ferussac)]

The influences of two water soluble contrast media, meglumine iothalamate and meglumine iocarmate, on the neuronal excitability and on the neuronal sensitivity to putative transmitters were examined in comparison with those of sucrose using two identifiable giant neurones of Achatina fulica Férussac (the TAN and the PON). A relatively low increase of osmotic pressure of the extracellular fluid, produced by the application of contrast media, reversed the Cl- dependent inhibition caused by a putative transmitter. The same increase of this osmotic pressure, however, did not influence the Cl- independent inhibition and the excitation of the neurone examined. The hyperpolarization of neuromembrane was caused by an increase of osmotic pressure of the extracellular fluid. Its relatively high increase was necessary to make spontaneous spike discharges disappear totally. All effects of the two contrast media, observed in this study, were due to the increase of osmotic pressure of the extracellular fluid ; no specific effect of the contrast media containing the iodine on the indicators used was observed.     

Structure-activity relationship studies on the endogenous neuroactive tetrapeptide achatin-I on giant neurons of Achatina fulica Ferussac.

The structure-activity relationships of achatin-I, a neuroactive peptide containing a D-phenylalanine residue, for producing excitatory effects on three different types of Achatina neurons, PON, TAN and d-RCDN, were studied under the voltage clamp method. Of the peptides examined, only Gly-Gly-D-Phe-L-Ala-L-Asp (IV). D-Phe-L-Ala-L-Asp (V) and Gly-D-Phe-L-Ala-L-Asn (XVI) produced an inward current with increased membrane conductance similar to achatin-1 (I). The structure-activity relationship was essentially the same for the three Achatina neuron types. The equiactive molar ratios (EMRs) of the active peptides vs. achatin-I (I) were calculated from their dose-response curves: 8 - 60 for Gly-Gly-DPhe-L-Ala-L-Asp (IV), 200 - greater than 250 for D-Phe-L-Ala-L-Asp (V) and greater than 200 for Gly-D-Phe-L-Ala-L-Asn (XVI). These values indicate that the achatin-I receptor in the Achatina neurons is highly structure-specific.     

Effects of synthetic peptides on giant neurones identified in the ganglia of an African giant snail (Achatina fulica Férussac). IV

1. The previous papers (Ku et al., 1986; Kim et al., 1987; Yongsiri et al., 1987) reported the effects of the synthetic peptides, i.e. Met-enkephalin, substance P, neurotensin, oxytocin, Arg-vasopressin, proctolin, FMRFamide, ranatensin C etc., on about 20 identifiable giant neurones of an African giant snail (Achatina fulica Férussac). 2. In the present study, the effects of the same peptides on the following Achatina neurones, other than those of the previous papers, were investigated: v-RPLN, v-LPSN, v-VNAN, v-VLN, r-VMN, l-VMN, v-l-VOrN and d-RCDN. 3. Of the neurones tested here, v-RPLN (ventral-right parietal large neurone) was excited slightly by Met-enkephalin, excited markedly by oxytocin, and inhibited by FMRFamide, at 10(-4) M. 4. Of these effects, those of oxytocin and FMRFamide were undoubtedly the direct effects on the neurone tested, whereas those of Met-enkephalin were probably due to the synaptic activations. 5. Another neurone, v-LPSN (ventral-left parietal large neurone), was affected by oxytocin and ranatensin C at 10(-4) M. The two substances sometimes showed similar simple excitatory effects, in other cases biphasic (excitation followed by inhibition) effects, and in a few cases almost no effect. 6. The rest of the neurones tested were not sensitive at all to any of the peptides examined.     

Different effects of the replacement of extracellular sodium with lithium on the abnormal spike discharges caused by 2 convulsants, Metrazol and strychnine, on an identifiable giant neuron of the mollusc Achatina fulica Ferussac

The influences of the replacement of sodium with lithium in the extracellular medium on the abnormal spike discharges, caused by two convulsants, metrazol and strychnine, of a giant neurone (TAN, tonically autoactive neurone) identified in the suboesophageal ganglia of the African giant snail (Achatina fulica Férussac) were examined. The slow oscillations of potential caused by metrazol disappeared after this replacement. On the other hand, the abnormal action potentials caused by strychnine, as well as the normal action potentials, still remained after the removal of sodium in the medium.     

Effects of synthetic biologically active peptides on giant neurones identified in the left buccal ganglion of an African giant snail (Achatina fulica Férussac)

1. The effects of synthetic biologically active peptides, including Met-enkephalin, substance P, oxytocin, Arg-vasopressin, proctolin and FMRFamide, on the following four buccal neurones were examined: d-LBAN (dorsal-left buccal anterior neurone), d-LBMN (dorsal-left buccal medial neurone), d-LBCN (dorsal-left buccal central neurone) and d-LBPN (dorsal-left buccal posterior neurone). These peptides were examined at 10(-4) M. 2. Oxytocin excited d-LBAN and slightly excited d-LBCN, while this inhibited d-LBMN. Arg-vasopressin excited slightly d-LBAN and d-LBCN, but this had some times no effect. FMRFamide inhibited d-LBAN, and slightly inhibited d-LBCN. 3. No direct synaptic connection from the two ventral cerebral giant neurones, v-LCDN and v-RCDN, to the four buccal giant neurones was found, though the two cerebral neurones innervate the cerebro-buccal connectives.     

Isolation, amino acid sequence, synthesis, and biological activity of some oligopeptides from porcine spinal cord

Four oligopeptides, designated SCP-3, SCP-4, SCP-5, and SCP-6, have been isolated and purified to homogeneity from porcine spinal cord. The amino acid sequences have been determined as pyroGlu-Gly, pyroGlu-Gly-Gly, Met-Met-Gly, and Asp-Ala-Gly-Ala-Gly, respectively. All of these peptides have been synthesized by conventional liquid-phase or solid-phase methods. The synthetic and extracted peptides showed identical behavior in a reverse-phase high-performance liquid chromatography system. SCP-3 and SCP-4 exhibited some significant inhibitory activity on the electrical stimulation-induced contractions of longitudinal muscle strip of guinea pig ileum, and SCP-5 showed some stimulating effect on the same preparation. The physiological significance of these purified peptides is being investigated.     

The actions of rfamide neuroactive peptides on the isolated heart of the giant african snail,Achatina fulica

1. The effects of a range of RFamide peptides were examined on the frequency, amplitude and tone of the isolated heart of Achatina fulica.2. FMRFamide, FLRFamide and SDPNFLRFamide were potent excitants while LPLRFamide, KHEYLRFamide and GSFFRFamide were weak excitants.3. DNFLRFamide and SSLFRFamide were potent inhibitory peptides while LFRFamide, GSLFRFamide and KNEFIRFamide were weak inhibitory peptides.4. MRFamide, LRFamide and RFamide were inactive.5. It is concluded that a tetrapeptide sequence is the minimal requirement for activity on the Achatina heart. With the exceptions of DNFLRFamide and in most preparations GSFFRFamide, LRFamide peptides are excitatory while FRFamide peptides are inhibitory.6. Due to its inhibitory effect and high potency, the sequence DNFLRFamide warrants further investigation.     

Potential of Plant’s Dipeptidyl Peptidase I & II Homologs in Generation of ACE Inhibitory Peptides

Synthetic inhibitors of angiotensin converting enzyme (ACE) are commonly used in the treatment of hypertension and other cardiovascular diseases. But their diverse side effects stipulated nontoxic safer and economic inhibitors which can be accomplished by using peptidyl inhibitors from natural sources or functional food ingradients. Dipeptidyl peptidases cleaved dipeptide moieties from amino terminus of oligopepides so, they may be used to generate effective dipeptidyl ACE inhibitors. In present study, role of purified DPP-I and II in generation of ACE inhibitors have been explored. Results showed that collagen alpha-1(III) from chicken showed highest ACE inhibitory potential. Both dipeptidyl peptidases hydrolysed various potent inhibitory dipeptides from their oligopeptide precursors. In addition, sequential digestion of proteins with trypsin, DPP-I and II released approximate 15 % of total inhibitory peptides. Furthermore, inhibitory peptide concentration can be increased up to 30 % or more by using more proteases in presence of DPP-I and II. Results revealed that both DPP-I and II possesses the ability to generate ACE dipeptide inhibitors from oligopeptides. Still various dipeptidyl inhibitory peptides remained in generating oligopeptides, which required study of other endopeptidases with broad specificities.     

Interaction of the human topoisomerase I-DNA complex with oligo-1,3-thiazolecarboxamides and their oligonucleotide conjugates

Nonnatural thiazole-containing oligopeptides (TCOs) bind to the DNA minor groove and inhibit the reaction catalyzed by human topoisomerase I (TopoI). The effect is directly proportional to the number of thiazole monomers in TCO. Several TCOs with three or four thiazole monomers act 3–10 times more efficiently than distamycin A, a natural antibiotic containing pyrrole rings. Additional groups at the N and C termini only slightly affect TopoI inhibition by TCO. The inhibitory effect of TCOs is higher than that of homo-or heterooligopeptides containing imidazole or pyrrole monomers, and the most potent are oligopeptide-oligonucleotide conjugates. The plausible causes of the different effects of distamycin and the nonnatural peptides on DNA relaxation catalyzed by TopoI are discussed.     

Effects of synthetic omega-conotoxin GVIA (omega-CgTX GVIA) on the membrane calcium current of an identifiable giant neurone, d-RPLN, of an African giant snail (Achatina fulica Ferussac), measured under the voltage clamp condition

1. The sodium and the calcium inward currents (INa and ICa) of an identifiable giant neurone, d-RPLN (dorsal-right parietal large neurone), of an African giant snail (Achatina fulica Férussac), were measured separately under the voltage clamp condition. 2. The effects of synthetic omega-conotoxin GVIA (omega-CgTX GVIA) on the calcium current of the neuromembrane were examined. 3. omega-CgTX GVIA is a peptide venom originally isolated from a fish-hunting marine snail (Conus geographus L.); the peptide venom has been demonstrated to block markedly calcium channels in vertebrates. 4. In the case of the d-RPLN membrane, the ICa was much larger than INa. The command voltage (Vc) to get the ICa in maximum was about 0 mV; the maximum value of ICa in a representative experimental case, was measured as approximately 0.8 microA. 5. With respect to the ICa of a molluscan giant neurone, d-RPLN, synthetic omega-CgTX GVIA at a high concentration, 5 X 10(-5) M, showed almost no effect, in spite of reporting the peptide venom affecting the ICa in vertebrate preparations.     

Thoracic output of crayfish giant fibres

Summary The thoracic homologue of the abdominal segmental giant neurone of crayfish Pacifastacus leniusculus is identified and described. It has a small cell body located in the anterior ventro-lateral quadrant of the ganglion and a large neuropil arborization, with dendrites aligned along the tracts of the giant fibres. The SG axon exits the ganglion within the major root which innervates the leg, usually in the anterior region of this root. Within 1–2 mm of the ganglion the axon terminates in a mass of fine branches, apparently randomly located within the base of the root.The SG receives suprathreshold input from the ipsilateral MG and LG fibres through rectifying electrical synapses. It makes output to FF motor neurones, also through electrical synapses. The SG also makes output to at least one corollary discharge interneurone. The SG receives depolarizing inhibitory synaptic potentials which can prevent its activation by the GFs. Some but not all of these synaptic potentials are common to similar potentials occurring in a large leg promotor motor neurone.Abbreviations AC anterior connective - GF giant fibre - IPSP inhibitory post-synaptic potential - LG lateral giant fibre - MG medial giant fibre - MoG motor giant neurone - PC posterior connective - PMM promotor motor neurone - r1 first root - r3 third root - rAD anterior distal root - rPD posterior distal root - rPM promotor muscle root - SG segmental giant neurone