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 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-NH2 ([Ser2]MIP) and H-Gly-Ala-Pro-Met-Phe-Val-NH2 ([Ala2]MIP), and their fragments, H-Pro-Met-Phe-Val-NH2 (MIP-(3-6)) and H-Met-Phe-Val-NH2 (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-NH2 ([Ala2, Val5, Phe6]MIP-NH2) and H-Gly-Ala-Pro-Met-NH2 ([Ala2]MIP-(1-4)-NH2), were ineffective. The potency order of the four effective peptides was determined from their dose-response relations: [Ser2]MIP greater than [Ala2]MIP greater than MIP-(3-6) greater than MIP-(4-6). The ED50 of [Ser2]MIP was about 3 X 10(-5) M. Relations between the outward current (nA) and the conductance increase (microseconds) produced by the four peptides were quite linear (Y = 0.03416 X-0.01083, r = 0.98677). The reversal potentials for [Ser2]MIP (ES-MIP) measured with various extracellular K+ concentrations ([K+]0) were fitted to the Nernst equation, being identical with EK. ES-MIP was not affected by changing [Na+]0 and [Cl-]0.

Achatin-I, an endogenous neuroexcitatory tetrapeptide from Achatina fulica Férussac containing a D-amino acid residue.

A tetrapeptide named achatin-I was purified from the suboesophageal and cerebral ganglia of the African giant snail Achatina fulica Férussac, and evoked a potent neuroexcitatory effect. The amino acid sequence of achatin-I is Gly-D-Phe-Ala-Asp. Achatin-I induced a voltage-dependent inward current, due to Na+, on the identifiable giant neuron, periodically oscillating neuron (PON), of the same snail. All possible isomers of achatin-I were synthesized using the solid-phase method. The sensitivity of the neuron to achatin-I and its isomers was strictly stereospecific; among the various isomers, only achatin-I showed marked effects (ED50 = 2.29 x 10(-6)M), while Gly-D-Phe-D-Ala-Asp, the synthetic D-Ala-isomer, was less than 10(-3) active.

Classification of GABA receptors in snail neurones.

The present study aimed to elucidate the pharmacological features of GABA receptors on identifiable neurones of Achatina fulica Férussac by testing the effects of GABA analogues, muscimol, (+/-)-baclofen, (-)-beta-hydroxy GABA and those conformationally fixed in either the extended or folded form of carbon chain, such as trans- and cis-isomers of (+/-)-2-(aminomethyl)cyclopropane-1-carboxylic acid [+/-)-cyclo-GABA-extended and (+/-)-cyclo-GABA-folded) and trans-4-amino-crotonic acid (GABA-extended). The giant neurones used were TAN, d-LPeLN, v-VNAN, v-LCDN and RPeNLN. The minimum effective concentrations (MEC) of these compounds to produce hyper- or depolarization of the membrane potentials of the neurones were determined, and the effective potency quotient (EPQ) of each compound vis-à-vis that of GABA was calculated for each neurone. The GABA receptors in these neurones were classified into the muscimol I, muscimol II and baclofen types. The muscimol I (TAN and d-LPeLN) and muscimol II (v-VNAN and v-LCDN) receptors were respectively hyperpolarized and depolarized by GABA and muscimol but were insensitive to (+/-)-baclofen. These muscimol receptors are inferred to accept GABA in an extended form of its carbon chain, since muscimol, conformationally fixed in this form from C-1 to C-4, was quite effective. Muscimol was more potent on the muscimol II receptors (MEC: 3 X 10(-7)-3 X 10(-6) M; EPQ: 30-10) than on the muscimol I type (MEC: 3 X 10(-5)-10(-4) M; EPQ: 1-0.3).(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Effects of synthetic peptides on giant neurons identified in the ganglia of an African giant snail (Achatina fulica Férussac)--II.

Thirteen synthetic biologically-active peptides, which were classified into the peptides proposed as neurotransmitters in mammals and invertebrates and neural venom peptides, were investigated for their effects on the following six identifiable giant neurons of an African giant snail (Achatina fulica Férussac): RAPN (right anterior pallial neuron), INN (intestinal nerve neuron), RPeNLN (right pedal nerve large neuron), LPeNLN (left pedal nerve large neuron), d-LPeLN (dorsal-left pedal large neuron) and d-LPeCN (dorsal-left pedal constantly firing neuron). Oxytocin and proctolin at 10(-4)M excited the RAPN membrane potential, whereas FMRFamide at the same concentration inhibited the same neuron. FMRFamide at 10(-4)M markedly inhibited the d-LPeLN membrane potential, sometimes produced inhibition of RPeNLN and LPeNLN, showed varied effects (excitatory or inhibitory) on INN, and had no effect on d-LPeCN. The other peptides examined had almost no effect on any of the neurons tested.

Histological and pharmacological investigations of the two symmetrically situated giant neurons, RPeNLN and LPeNLN, identified on the anterior surface of the pedal ganglia of an African giant snail (Achatina fulica Férussac).

Morphological and pharmacological investigations were made of two giant neurons, RPeNLN (right pedal nerve large neuron) and LPeNLN (left pedal nerve large neuron), situated symmetrically on the anterior surface of the pedal ganglia of an African giant snail (Achatina fulica Férussac). The two neurons (about 250-300 microns in diameter) were the largest ones identified in the ganglia of the snail species. The axonal pathways of the two neurons were symmetrical; of their four main axonal branches, the three main branches innervated the ipsilateral pedal nerves, whereas the last main branch projected to the contralateral pedal nerves. The pharmacological features of the two neurons were very similar. Both were inhibited markedly by dopamine [minimum effective concentrations (MECs): 3 X 10(-6)-10(-5) M], DL-octopamine (MECs: 2 X 10(-6)-2 X 10(-5) M), 5-hydroxytryptamine (MEC: 3 X 10(-6) M), GABA (MEC: 3 X 10(-4) M), L-homocysteic acid (MECs: 3 X 10(-5)-10(-4) M) and erythro-beta-hydroxy-L-glutamic acid (MEC: 3 X 10(-5) M). Acetylcholine showed varied effects, either excitatory or inhibitory, on the two neurons examined. No substances were found to have any marked excitatory effects on the neurons.