Identification of a tachykinin-related neuropeptide from the honeybee brain using direct MALDI-TOF MS and its gene expression in worker, queen and drone heads.

Using a combination of MALDI-TOF and on-line capillary HPLC/Q-Tof mass spectroscopy, we identified and determined the amino acid sequence of a novel neuropeptide in the brain of the honeybee Apis mellifera L., termed AmTRP peptide (Apis mellifera tachykinin-related peptide), related to insect tachykinin. A cDNA for a prepro-protein (prepro-AmTRP) of AmTRP was isolated and determined to encode seven AmTRPs 1-7. Northern blot analysis indicated that the prepro-AmTRP gene is expressed differentially in the nurse bee, forager, queen and drone heads. Strong expression was detected in the queen and forager heads, while weak and almost no significant expression was detected in the nurse and drone heads, respectively. These results suggest that AmTRP peptide functions as a neuromodulator and/or hormone, associated with sex-specific or age/division of labour-selective behaviour and/or physiology of the honeybees.

Differing effects of substrate and non-substrate transport inhibitors on glutamate uptake reversal.

Na(+)-dependent excitatory amino acid transporters (EAATs) normally function to remove extracellular glutamate from brain extracellular space, but EAATs can also increase extracellular glutamate by reversal of uptake. Effects of inhibitors on EAATs can be complex, depending on cell type, whether conditions favor glutamate uptake or uptake reversal and whether the inhibitor itself is a substrate for the transporters. The present study assessed EAAT inhibitors for their ability to inhibit glutamate uptake, act as transporter substrates and block uptake reversal in astrocyte and neuron cultures. L-threo-beta-hydroxyaspartate (L-TBHA), DL-threo-beta-benzyloxyaspartate (DL-TBOA), L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans-2,4-PDC) (+/-)-cis-4-methy-trans-pyrrolidine-2,4-dicarboxylic acid (cis-4-methy-trans-2,4-PDC) and L-antiendo-3,4-methanopyrrolidine-2,4-dicarboxylic acid (L-antiendo-3,4-MPDC) inhibited L-[14C]glutamate uptake in astrocytes with equilibrium binding constants ranging from 17 microM (DL-TBOA and L-TBHA) - 43 microM (cis-4-methy-trans-2,4-PDC). Transportability of inhibitors was assessed in astrocytes and neurons. While L-TBHA, L-trans-2,4-PDC, cis-4-methy-trans-2,4-PDC and L-antiendo-3,4-MPDC displayed significant transporter substrate activities in neurons and astrocytes, DL-TBOA was a substrate only in astrocytes. This effect of DL-TBOA was concentration-dependent, leading to complex effects on glutamate uptake reversal. At concentrations low enough to produce minimal DL-TBOA uptake velocity (< or = 10 microM), DL-TBOA blocked uptake reversal in ATP-depleted astrocytes; this blockade was negated at concentrations that drove substantial DL-TBOA uptake (> 10 microM). These findings indicate that the net effects of EAAT inhibitors can vary with cell type and exposure conditions.

Effects of threo-beta-hydroxyaspartate derivatives on excitatory amino acid transporters (EAAT4 and EAAT5).

D,L-threo-beta-Benzyloxyaspartate (D,L-TBOA), an analog of threo-beta-hydroxyaspartate (THA) possessing a bulky substituent, is a potent non-transportable blocker for the excitatory amino acid transporters, EAAT1, 2 and 3, while L-threo-beta-methoxyaspartate (L-TMOA) is a blocker for EAAT2, but a substrate for EAAT1 and EAAT3. To characterize the actions of these THA analogs and the function of EAAT4 and EAAT5, we performed electrophysiological analyses in EAAT4 or EAAT5 expressed on Xenopus oocytes. In EAAT4-expressing oocytes, D,L-TBOA acted as a non-transportable blocker, while L-TMOA like D,L-THA was a competitive substrate. In contrast, D,L-THA, D,L-TBOA and L-TMOA all strongly attenuated the glutamate-induced currents generated by EAAT5. Among them, L-TMOA showed the most potent inhibitory action. Moreover, D,L-THA, D,L-TBOA and L-TMOA themselves elicited outward currents at negative potentials and remained inward at positive potentials suggesting that D,L-TBOA and L-TMOA, as well as D,L-THA, not only act as non-transportable blockers, but also block the EAAT5 leak currents. These results indicate that EAATs 4 and 5 show different sensitivities to THA analogs although they share properties of a glutamate-gated chloride channel.

Syntheses of optically pure beta-hydroxyaspartate derivatives as glutamate transporter blockers.

DL-threo-beta-benzyloxyaspartate (DL-TBOA) is a non-transportable blocker of the glutamate transporters that serves as an indispensable tool for the investigation of the physiological roles of the transporters. To examine the precise interaction between a blocker and the transporters, we synthesized the optically pure isomers (L- and D-TBOA) and its erythro-isomers. L-TBOA is the most potent blocker for the human excitatory amino acid transporters (EAAT1-3), while D-TBOA revealed a difference in the pharmacophores between EAAT1 and EAAT3. We also synthesized the substituent variants (methyl or naphthylmethyl derivatives) of L-TBOA. The results obtained here suggest that bulky substituents are crucial for non-transportable blockers.

Identification of orcokinin gene-related peptides in the brain of the crayfish Procambarus clarkii by the combination of MALDI-TOF and on-line capillary HPLC/Q-Tof mass spectrometries and molecular cloning.

We developed a strategy for the exploration of brain peptides in the red swamp crayfish, Procambarus clarkii, utilizing the combined techniques of matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry (MALDI-TOF MS), molecular cloning, and on-line capillary reversed-phase HPLC/quadrupole orthogonal acceleration time-of-flight (Q-Tof)-MS. We initially performed direct MALDI-TOF MS analysis with slices of the brain. The MS spectra from a slice of the olfactory lobe indicated that an orcokinin (NFDEIDRSGFGFN) occurs in this species. Subsequently, its occurrence was confirmed by molecular cloning of the cDNAs encoding the precursor protein of orcokinin. The deduced amino acid sequences indicated that there are two different types of preproorcokinins. Preproorcokinin A (251 residues long) contains not only seven copies of orcokinin but also two copies of NFDEIDRSGFGFV and one copy each of NFDEIDRSGFGFA, NFDEIDRTGFGFH, and FDAFTTGFGHS. The former three peptides were previously isolated from another crayfish, Orconectes limosus, and/or the shore crab, Carcinus maenas, and the latter two were novel. Preproorcokinin B (266) harbors one additional orcokinin. All sequences of the peptides are flanked by dibasic sequences which are the consensus signal for processing. Moreover, brain extract was subjected to Sephadex G-25 and, subsequently, to on-line capillary reversed-phase HPLC/Q-Tof MS analysis. From the LC-MS analysis, the molecular weights of orcokinin, NFDEIDRSGFGFV, NFDEIDRSGFGFA, NFDEIDRTGFGFH, and FDAFTTGFGHS were identified as the doubly charged ions at m/z 759.37, 751.92, 737.86, 777.90, and 593. 78, respectively. In addition, the sequences were assigned by the collision-induced dissociation spectra using the doubly charged ions in the LC-MS/MS analysis. These data suggest that orcokinin and its related peptides are especially abundant in the olfactory lobe and are synthesized and processed from the two types of preproorcokinins in the crayfish brain.

Inhibition of uptake unmasks rapid extracellular turnover of glutamate of nonvesicular origin.

Maintaining glutamate at low extracellular concentrations in the central nervous system is necessary to protect neurons from excitotoxic injury and to ensure a high signal-to-noise ratio for glutamatergic synaptic transmission. We have used DL-threo-beta-benzyloxyaspartate (TBOA), an inhibitor of glutamate uptake, to determine the role of glutamate transporters in the regulation of extracellular glutamate concentration. By using the N-methyl-D-aspartate receptors of patched CA3 hippocampal neurons as "glutamate sensors," we observed that application of TBOA onto organotypic hippocampal slices led to a rapid increase in extracellular glutamate concentration. This increase was Ca(2+)-independent and was observed in the presence of tetrodotoxin. Moreover, prevention of vesicular glutamate release with clostridial toxins did not affect the accumulation of glutamate when uptake was inhibited. Inhibition of glutamine synthase, however, increased the rate of accumulation of extracellular glutamate, indicating that glial glutamate stores can serve as a source in this process. TBOA blocked synaptically evoked transporter currents in astrocytes without inducing a current mediated by the glutamate transporter. This indicates that this inhibitor is not transportable and does not release glutamate by heteroexchange. These results show that under basal conditions, the activity of glutamate transporters compensates for the continuous, nonvesicular release of glutamate from the intracellular compartment. As a consequence, acute disruption of transporter activity immediately results in significant accumulation of extracellular glutamate.

Characterization of a cDNA encoding a precursor polypeptide of a D-amino acid-containing peptide, achatin-I and localized expression of the achatin-I and fulicin genes.

Achatin-I and fulicin, isolated from the ganglia and atria of the giant land snail Achatina fulica, are a tetrapeptide and pentapeptide containing a d-Phe and d-Asn at position 2, respectively. We succeeded in cloning a cDNA encoding a precursor of achatin-I from the Achatina ganglia, revealing that the d-Phe present in achatin-I is coded by a common l-Phe codon, TTT or TTC. The deduced polypeptide was found to comprise seven repeats of the achatin sequence GFAD and one analog GFGD flanked on both sides by the typical endoproteolytic site KR. Northern blot analysis of transcripts and Southern blot analysis of reverse transcription (RT)-PCR products demonstrated that achatin-I mRNA was localized in the subesophageal ganglia, whereas expression of fulicin mRNA was detected in the atrium as well as in the subesophageal ganglia. Furthermore, localization of the achatin gene transcript in the right and left pedal ganglia compartments was shown by in situ hybridization on sections of subesophageal ganglia, whereas the fulicin transcript was observed in the right and left parietal ganglia. These data suggested that achatin-I plays an essential role in the regulation of the heart as a neurotransmitter or neurohormone through production in the pedal ganglia and transport to the atrium, whereas fulicin serves not only as a neurotransmitter or neurohormone but also as a novel atrial hormone.

DL-threo-beta-benzyloxyaspartate, a potent blocker of excitatory amino acid transporters.

DL-threo-beta-Benzyloxyaspartate (DL-TBOA), a novel derivative of DL-threo-beta-hydroxyaspartate, was synthesized and examined as an inhibitor of sodium-dependent glutamate/aspartate (excitatory amino acid) transporters. DL-TBOA inhibited the uptake of [14C]glutamate in COS-1 cells expressing the human excitatory amino acid transporter-1 (EAAT1) (Ki = 42 microM) with almost the same potency as DL-threo-beta-hydroxyaspartate (Ki = 58 microM). With regard to the human excitatory amino acid transporter-2 (EAAT2), the inhibitory effect of DL-TBOA (Ki = 5.7 microM) was much more potent than that of dihydrokainate (Ki = 79 microM), which is well known as a selective blocker of this subtype. Electrophysiologically, DL-TBOA induced no detectable inward currents in Xenopus laevis oocytes expressing human EAAT1 or EAAT2. However, it significantly reduced the glutamate-induced currents, indicating the prevention of transport. The dose-response curve of glutamate was shifted by adding DL-TBOA without a significant change in the maximum current. The Kb values for human EAAT1 and EAAT2 expressed in X. laevis oocytes were 9.0 microM and 116 nM, respectively. These results demonstrated that DL-TBOA is, so far, the most potent competitive blocker of glutamate transporters. DL-TBOA did not show any significant effects on either the ionotropic or metabotropic glutamate receptors. Moreover, DL-TBOA is chemically much more stable than its benzoyl analog, a previously reported blocker of excitatory amino acid transporters; therefore, DL-TBOA should be a useful tool for investigating the physiological roles of transporters.

Molluscan neuropeptides.

Achatin-I, fulicin, fulyal, Mytilus-FFRFamide and Helix CCAP-RP-III are D-amino acid-containing neuropeptides from molluscs. Achatin-I, fulicin and fulyal from Achatina showed excitatory and/or modulatory actions on the penis retractor, radula retractor or ventricular muscles and neurons, though their L isomers were devoid of activity. On the other hand, both Mytilus-FFRFamide and its L isomer showed excitatory effects on the anterior byssus retractor muscle. Moreover, in contrast to Achatina neuropeptides, Helix CCAP-RP-III exhibited no remarkable activities on any of the muscles tested; instead, its L isomer possessed various excitatory effects. The molecular structures of these short peptides would be affected by the L-->D conversion and could influence activity. Molecular biological studies on the fulicin precursor suggest that fulicin, fulyal and related peptides are produced in Achatina ganglia and heart by processing of the ribosomally made precursor, and that L-isomeric fulicin and fulyal further undergo epimerization to yield the D-isomers.

New beta-hydroxyaspartate derivatives are competitive blockers for the bovine glutamate/aspartate transporter.

Four subtypes of excitatory amino acid transporters (EAAT1-4) have been identified in the mammalian brain. A number of pharmacological agents have been developed to study their intrinsic properties and function. Up to now, blockers were available only for EAAT2, whereas all the inhibitors of glutamate uptake active on the other subtypes were proved to be substrates of the transporters. We synthesized five new derivatives of DL-threo-beta-hydroxyaspartic acid, a well known general substrate of EAATs, and investigated their potential blocking activity on the cloned bovine EAAT1 expressed in the Xenopus oocyte system, by using radiotracer and voltage-clamp techniques. Two of our derivatives proved to be substrates for bovine EAAT1, with reduced electrogenicity compared with their parent compound, and an affinity of 40 and 64 microM. The last three derivatives displayed a blocking activity on bovine EAAT1. The affinity of DL-threo-beta-benzoyloxyaspartate and DL-threo-beta-(1-naphthoyl)oxyaspartate was determined by Schild analysis as 17.2 and 52.1 microM, respectively. These blockers should help in the better understanding of the key intrinsic properties of EAAT1. Moreover, they appear as good candidates for a general blocking activity on EAATs.

A novel D-amino acid-containing peptide, fulyal, coexists with fulicin gene-related peptides in Achatina atria.

Fulicin (Phe-D-Asn-Glu-Phe-Val-NH2) is a neuropeptide from ganglia of the African giant snail (Achatina fulica). Previously, the sequences of nine fulicin gene-related peptides (FGRP-1 to -9) have been predicted from the cDNA encoding the ganglia fulicin precursor and the transcripts have been detectable in the heart. We synthesized twenty peptides related to fulicin and FGRPs containing either an L- or a D-amino acid at position 2 and used them to identify FGRPs in atrial extracts. We identified ten alpha-amidated peptides, including fulicin and confirmed their structures as follows: Tyr-Ala-Glu-Phe-Leu-NH2 (FGRP-9), [D-Ala2]FGRP-9 (fulyal), [L-Asn2]fulicin, fulicin, Ser-Tyr-Asp-Phe-Val-NH2 (FGRP-2), Thr-Tyr-Asp-Phe-Leu-NH2 (FGRP-3), Tyr-Asp-Phe-Ile-NH2 (FGRP-5), Ser-Pro-Tyr-Asp-Phe-Ile-NH2 (FGRP-6), Asn-Tyr-Asp-Phe-Val-NH2 (FGRP-7) and Ser-Pro-Tyr-Asp-Phe-Val-NH2 (FGRP-8). We analyzed the biological activities of synthetic FGRPs using the snail penis retractor muscle. The results revealed that fulyal remarkably potentiated the tetanic contraction at concentrations as low as 10(-12) M. FGRP-9 was about 10,000-fold less potent. Fulyal, like fulicin, seems to undergo preferential maturation to participate in the penis retractor muscle contraction as a neuropeptide containing a D-amino acid.

A novel cDNA sequence encoding the precursor of the D-amino acid-containing neuropeptide fulicin and multiple alpha-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.

A Mytilus peptide related to the small cardioactive peptides (SCPs): structure determination and pharmacological characterization.

1. An SCP-related peptide, Ala-Pro-Asn-Phe-Leu-Ala-Tyr-Pro-Arg-Leu-NH2, was isolated from the ABRMs of Mytilus edulis. The peptide was designated Mytilus SCP. 2. At 10(-10) M or higher, Mytilus SCP showed a potentiating effect on phasic contraction of the ABRM in response to repetitive electrical stimulation. In contrast, the peptide did not show any potentiating effect on contractures in response to ACh and the FMRFamide-related Mytilus decapeptide, suggesting that the potentiating effect on phasic contraction was brought about by an action of the peptide on the nerve elements in the ABRM. 3. At 10(-8) M or higher, Mytilus SCP showed a catch-relaxing effect in addition to the potentiating effect. The relaxing effect was blocked by mersalyl, suggesting that it was also brought about by a presynaptic action. 4. Various analogues of Mytilus SCP were examined on the ABRM. Leu-Ala-Tyr-Pro-Arg-Leu-NH2 was suggested to be the minimum structure required for both potentiating and relaxing activities on the ABRM. Leu-D-Ala-Tyr-Pro-Arg-D-Leu-NH2 and D-Leu-Ala-Tyr-Pro-Arg-D-Leu-NH2 were found to show strong potentiating and relaxing activities, though they were less potent than the Mytilus SCP.

Effect of the D-Phe2 residue on molecular conformation of an endogenous neuropeptide achatin-I. Comparison of X-ray crystal structures of achatin-I (H-Gly-D-Phe-Ala-Asp-OH) and achatin-II (H-Gly-Phe-Ala-Asp-OH).

The molecular conformation of achatin-II neutral form (H-Gly-Phe-Ala-Asp-OH), an endogenous peptide from the Achatina fulica ganglia, was elucidated by X-ray crystal analysis. The molecule takes an extended beta-pleated structure stabilized by 5 intermolecular hydrogen bonds with the antiparallely arranged molecules. This is in contrast with the turn conformation of a neuroactive achatin-I (H-Gly-D-Phe-Ala-Asp-OH) [(1992) FEBS Lett. 276,95-97]. The conformational comparison of both of the molecules makes clear the structural role which D-Phe residue of achatin-I plays in forming a definite active form.

The FMRFamide-related decapeptide of Mytilus contains a D-amino acid residue.

1. An FMRFamide-related decapeptide isolated from the anterior byssus retractor muscle (ABRM) of the bivalve mollusc, Mytilus edulis, was shown to have D-Leu as the second amino acid residue. 2. The excitatory effects of the peptide (Mytilus-FFRFamide) on the ABRM were not changed appreciably by substituting an L-Leu residue for the D-Leu residue.

Trypsin inhibitors from bottle gourd (Lagenaria leucantha Rusby var. Depressa Makino) seeds. Purification and amino acid sequences.

Two almost identical trypsin isoinhibitors, LLDTI-I and LLDTI-II, from bottle gourd (Lagenaria leucantha Rusby var. Depressa Makino) seeds were purified by acetone precipitation, gel filtration and reversed phase chromatography. LLDTI-I and LLDTI-II consist of 30 and 29 amino acid residues, respectively, and have identical sequences, except that LLDTI-I has one additional pyroglutamic acid residue at N-terminus. Both proteins are strong inhibitors of bovine trypsin, with Ki values of 2.4.10(-10) M (LLDTI-I) and 9.6.10(-11) M (LLDTI-II). Amino acid sequences are as follows: [sequence: see text]

Crystal structure and molecular conformation of achatin-I (H-Gly-D-Phe-Ala-Asp-OH), an endogenous neuropeptide containing a D-amino acid residue.

In order to investigate the active conformation of achatin-I (H-Gly-D-Phe-Ala-Asp-OH), an endogenous neuropeptide from the Achatina fulica ganglia, its crystal structure and molecular conformation were analysed by the X-ray diffraction method. Crystals from methanol/dioxane are monoclinic, space group P2(1) with a = 5.083(1), b = 9.125(1), c = 20.939(3) A, beta = 94.73(1) degrees. The structure was solved by direct methods and refined to R = 0.051 for 1714 independent reflections with /Fo/ greater than sigma (Fo). The molecule exists as a zwitterion with the Gly N-terminal end protonated and Asp beta-carboxyl deprotonated; the C-terminal of Asp is in a neutral state. The molecule takes a kind of beta turn structure with the D-Phe-Ala residues at the corner of the bend. This turn conformation is primarily formed by the strong intramolecular hydrogen bonds of NH(Gly)...O delta 1 (Asp) and NH(Asp)...O delta 1 (Asp) pairs, thus forming a 15-membered ring structure. Judging from the published data concerning the structure-activity relationship, this turn conformation may reflect an important feature related to the neuroexcitatory activity of achatin-I.

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-NH2 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-.

Achatin-I, an excitatory neurotransmitter having a D-phenylalanine residue of Achatina giant neurones.

The neuroexcitatory peptide isolated from Achatina ganglia was identical to the synthetic Gly-D-Phe-L-Ala-L-Asp with respect to either the bioassay experiments using the Achatina neurones or the instrumental analysis (1H-NMR, SIMS, CD and HPLC). We termed it achatin-I (yield: 50 micrograms from 30,000 animals). Its stereoisomer, Gly-L-Phe-L-Ala-L-Asp, termed achatin-II, was also isolated from the ganglia (yield: 17 micrograms), but this was ineffective on the Achatina neurones. Of the eight possible stereoisomers, only achatin-I markedly showed excitatory effects on the two Achatina neurones, PON and TAN, and [D-Ala3] achatin-I (Gly-D-Phe-D-Ala-L-Asp) had the slight effects. Among the fourteen neurones tested, seven, including the two mentioned above, were excited by achatin-I, whereas no neurone was inhibited. Achatin-I produced an inward current (Iin) with an increase in the membrane conductance (g) under voltage clamp. ED50 of achatin-I for exciting the neurones were 0.20-1.47 x 10(-5) M, and its Emax were 6.33-5.02 nA. Of the achatin-I analogues examined, only the three, Gly-Gly-D-Phe-L-Ala-L-Asp, D-Phe-L-Ala-L-Asp and Gly-D-Phe-L-Ala-L-Asn, produced Iin, but much smaller than that of achatin-I. The equiactive molar ratios (EMRs) of the four effective related peptides (three analogues and a stereoisomer) vs. achatin-I were: 8-60 for Gly-Gly-D-Phe-L-Ala-L-Asp, 200 - > 250 for D-Phe-L-Ala-L-Asp and > 200 for Gly-D-Phe-L-Ala-L-Asn and Gly-D-Phe-D-Ala-L-Asp. The Iin induced by achatin-I was blocked under the /Na+/0-free state, but unaffected under the [Ca2+]-free (replaced with Co2+), [Cl-]0-free or [K+]-enriched (3.0 x) medium, indicating that the Iin is produced by the gNa increase of neuromembrane. We propose that achatin-I having a D-phenylalanine residue is an excitatory neurotransmitter of the Achatina neurones.