Molecular cloning of precursors for TEP-1 and TEP-2: The GGNG peptide-related peptides of a prosobranch gastropod, Thais clavigera.

TEP (Thais excitatory peptide)-1 and TEP-2 are molluscan counterparts of annelidan GGNG-peptides, identified in a neogastropod, Thais clavigera (Morishita et al., 2006). We have cloned two cDNAs encoding TEP-1 and TEP-2 precursor protein, respectively, by the standard molecular cloning techniques. Predicted TEP-1 precursor protein consists of 161 amino acids, while predicted TEP-2 precursor protein has 118 amino acids. Only a single copy of TEP was found on the respective precursor. The semi-quantitative RT-PCR showed that expression of TEP-1 was high in sub-esophageal, pleural, pedal and visceral ganglia, while it was low in supra-esophageal ganglion. By contrast, expression level of TEP-2 was high in pedal and visceral ganglia. In situ hybridization visualized different subsets of TEP-1 and TEP-2 expressing neurons in Thais ganglia. For example, supra-esophageal ganglion contained many TEP-2 expressing neuron, but not TEP-1 expressing ones. These results suggest that expression of TEP-1 and TEP-2 is differently regulated in the Thais ganglia.

Molecular cloning of two distinct precursor genes of NdWFamide, a d-tryptophan-containing neuropeptide of the sea hare, Aplysia kurodai.

NdWFamide (NdWFa) is a D-tryptophan-containing cardioexcitatory neuropeptide in gastropod mollusks, such as Aplysia kurodai and Lymanea stagnalis. In this study, we have cloned two cDNA encoding distinct precursors for NdWFa from the abdominal ganglion of A. kurodai. One of the predicted precursor proteins consisted of 90 amino acids (NWF90), and the other consisted of 87 amino acids (NWF87). Both of the predicted precursor proteins have one NWFGKR sequence preceded by the N-terminal signal peptide. Sequential double staining by in situ hybridization (ISH) and immunostaining with anti-NdWFa antibody suggested that NdWFa-precursor and NdWFa peptide co-exist in neurons located in the right-upper quadrant region of the abdominal ganglion. In ISH, NWF90-specific signal and NWF87-specific one were found in different subsets of neurons in the abdominal ganglia of Aplysia. The expression level of NWF90 gene estimated by RT-PCR is much higher than that of NWF87 gene. These results suggest that NWF90 precursor is the major source of NdWFa in Aplysia ganglia.

Pharmacological characteristics of endokinin C/D-derived peptides in nociceptive and inflammatory processing in rats.

Endokinins designated from the human TAC4 gene consist of endokinin A, endokinin B, endokinin C (EKC) and endokinin D (EKD). EKC/D is a peptide using the common carboxyl-terminal in EKC and EKD and consists of 12 amino acids, and exerts antagonistic effects on the induction of scratching behavior by substance P (SP). Some of SP-preferring receptor antagonists have several d-tryptophan (d-Trp); however, the pharmacological effect of EKC/D-derived peptides with d-Trp remains to be solved. Therefore, to clarify the pharmacological characteristics of EKC/D-derived peptides, effects of pretreatment with these peptides on SP-induced scratching and thermal hyperalgesia, formalin-induced flinching and carrageenan-induced inflammation were evaluated. Intrathecal administration of [d-Trp(8)]-EKC/D and [d-Trp(10)]-EKC/D showed a markedly long inhibitory effect, at least 14 h, whereas the antagonistic effects of [d-Trp(8,10)]-EKC/D and EKC/D without d-Trp disappeared after 1h. Furthermore, the inhibitory effect of [d-Trp(10)]-EKC/D-derived peptides was dependent on the number of amino acids from the amino-terminus, and the more numerous the amino acids, the more marked the antagonistic effect. Thus, these results indicate that the effective duration of EKC/D-derived peptides is dependent on the number of d-Trp in the carboxyl-terminal region and the amino-terminal region regulates the antagonistic effect of EKC/D.

Subcutaneous injection of endokinin C/D attenuates carrageenan-induced inflammation.

Endokinins, encoded by the human preprotachykinin C (PPT-C)/TAC4 gene, are peptides that consist of endokinin A (EKA), B (EKB), C (EKC) and D (EKD) and belong to the tachykinin family. Intrathecal injection of EKC/D (using the common carboxyl-terminal duodecapeptide in EKC and EKD) markedly attenuated the induction of thermal hyperalgesia and scratching behavior by intrathecal administration of substance P (SP), indicating that EKC/D has an antagonistic effect on the neurokinin 1 receptor (NK1R), SP-preferring receptor, at the spinal level; however, the pharmacological function of EKC/D at the periphery is not yet understood. Therefore, to clarify the effect of EKC/D on the peripheral tissue, the effect of subcutaneous injection of EKC/D on carrageenan-induced inflammation was examined. Subcutaneous injection of EKC/D attenuated an increase in paw volume following carrageenan-induced inflammation in a dose-dependent manner. Indeed, the increased paw volume was significantly decreased 40 min after treatment with 10(-4) M (10 nmol) and 10(-3) M (100 nmol) EKC/D (100 microl/rat). Similarly, injection of NK1R antagonists such as L-703,606 and Spantide I (10(-3) M) attenuated the increased paw volume following inflammation. Furthermore, the reduced withdrawal latency evoked by inflammation following subcutaneous injection of carrageenan was also dose-dependently attenuated by EKC/D administration. These results indicate that subcutaneous injection of EKC/D elicits an anti-inflammatory effect on carrageenan-induced inflammation.

Effect of the carboxyl-terminal of endokinins on SP-induced pain-related behavior.

The preprotachykinin C gene encodes four endokinins, A, B, C, and D. Endokinins A and B and substance P (SP) are typical tachykinin peptides since their carboxyl-terminal regions share an F-F-G-L-M-amide, while endokinins C and D share an F-Q-G-L-L-amide. It is demonstrated that pretreatment with a peptide consisting of a common sequence between endokinins C and D (EKC/D) attenuates the induction of scratching behavior and thermal hyperalgesia by intrathecal administration of SP or EKA/B (the carboxyl-terminal dacapeptide common in endokinins A and B), suggesting that leucine at the carboxyl-terminal of EKC/D may have a crucial role in eliciting these effects. When the effect of [Leu(11)]-SP and [Leu(10)]-EKA/B on SP-induced pain-related behavior was examined, the induction of pain-related behavior was markedly attenuated by pretreatment with these peptides. This indicates that leucine at the carboxyl-terminal of these peptides plays a crucial role in eliciting this antagonistic effect.

Leucine at the carboxyl-terminal of endokinins C and D contributes to elicitation of the antagonistic effect on substance P in rat pain processing.

Endokinins are tachykinin peptides designated from a human preprotachykinin C (PPT-C, TAC4) gene and consist of endokinin A (EKA), endokinin B (EKB), endokinin C (EKC) and endokinin D (EKD). A representative of mammalian tachykinins is substance P (SP), which functions as a neurotransmitter or modulator in the pain system; however, little is known about the role of these endokinins, especially EKC and EKD, in pain processing. Therefore, we evaluated the effects of EKC/D (using the common carboxyl-terminal duodecapeptide in EKC and EKD) on pain processing in rats. Pretreatment with EKC/D prevented induction of scratching behavior and thermal hyperalgesia by intrathecal administration of EKA/B (using the common C-terminal decapeptide in EKA and EKB) and SP and c-Fos expression in laminae I/II and V/VI of the spinal cord by noxious thermal stimulation. A prominent difference between EKC/D and SP is the presence of leucine instead of methionine at the carboxyl-terminal of EKC/D. Thus, to clarify whether leucine at the carboxyl-terminal of EKC/D plays an important role in determining the inhibitory effect of this peptide, we intrathecally administered [Met(12)]-EKC/D in which only leucine of EKC/D is replaced by methionine. This peptide did not exhibit the inhibitory effect on SP-induced scratching behavior or thermal hyperalgesia but conversely caused thermal hyperalgesia. Taken together, these findings indicate that EKC/D has an inhibitory effect on pain processing in the rat spinal cord, and the effect is due to leucine at the carboxyl-terminal of EKC/D.

Identification of a novel receptor for an invertebrate oxytocin/vasopressin superfamily peptide: molecular and functional evolution of the oxytocin/vasopressin superfamily.

Annetocin is structurally related to an OT (oxytocin)/VP (vasopressin) family peptide, which has been isolated from the earthworm Eisenia foetida and has been shown to induce OT-like egg-laying behaviour. We now report the identification of an endogenous AnR (annetocin receptor). The deduced AnR precursor displays high sequence similarity with OT/VP receptors. Genomic analysis of the AnR gene revealed that the intron-inserted position is conserved between the AnR gene and the mammalian OT/VP receptor genes. These results indicate that AnR and mammalian OT/VP receptors share a common ancestor gene. Administration of annetocin to the AnR expressed in Xenopus oocytes induced a calcium-dependent signal transduction. Reverse transcriptase-PCR analysis and in situ hybridization showed that the AnR gene is expressed specifically in the nephridia located in the clitellum region, although the nephridia are distributed throughout the worm body. This result suggests that annetocin induces egg-laying behaviour through its action on the nephridia. This is the first description concerning the functional correlation between an invertebrate OT/VP-related peptide and egg-laying behaviour.

Cloning of Octopus cephalotocin receptor, a member of the oxytocin/vasopressin superfamily.

We reported that the common octopus, Octopus vulgaris, in common with vertebrates, possesses two members of the oxytocin/vasopressin superfamily: octopressin (OP) and cephalotocin (CT). This was the first observation of its kind in invertebrates. As OP and CT have different biological activities, the presence of specific receptors has been proposed. We cloned the cDNA of an orphan receptor from Octopus brain and found it to encode a polypeptide of 397 amino acids that displays sequences characteristic of G-protein coupled receptors. The orphan receptor showed high homology to receptors of the oxytocin/vasopressin superfamily and seemed to conserve the agonist-binding pocket common to the oxytocin and vasopressin receptors. Xenopus oocytes that express the orphan receptor responded to the application of CT by an induction of membrane Cl(-) currents coupled to the inositol phosphate/Ca(2+) pathway. OP and the other members of the oxytocin/vasopressin superfamily did not activate this receptor. HPLC fractionation of the Octopus brain extract combined with an oocyte assay yielded a single substance that was identical to CT. On the basis of these results, we conclude that the cloned receptor is the CT receptor (CTR). Expression of CTR mRNA in Octopus was detected in the central and the peripheral nervous systems, the pancreas, the oviduct and the ovary. This receptor may mediate physiological functions of CT in Octopus such as neurotransmission, reproduction and metabolism.

Distribution of the Aplysia cardioexcitatory peptide, NdWFamide, in the central and peripheral nervous systems of Aplysia.

NdWFamide is an Aplysia cardioexcitatory tri-peptide containing D-tryptophan. To investigate the roles of this peptide, we examined the immunohistochemical distribution of NdWFamide-positive neurons in Aplysia tissues. All the ganglia of the central nervous system (CNS) contained NdWFamide-positive neurons. In particular, two left upper quadrant cells in the abdominal ganglion, and the anterior cells in the pleural ganglion showed extensive positive signals. NdWFamide-positive processes were observed in peripheral tissues, such as those of the cardio-vascular system, digestive tract, and sex-accessory organs, and in the connectives or neuropils in the CNS. NdWFamide-positive neurons were abundant in peripheral plexuses, such as the stomatogastric ring. To examine the NdWFamide contents of tissues, we fractionated peptidic extracts from the respective tissues by reversed-phase high-pressure liquid chromatography and then assayed the fractions by competitive enzyme-linked immunosorbent assay. A fraction corresponding to the retention time of synthetic NdWFamide contained the most immunoreactivity, indicating that the tissues contained NdWFamide. The prevalence of the NdWFamide content was roughly in the order: abdominal ganglion >heart >gill >blood vessels >digestive tract. In most of the tissues containing NdWFamide-positive nerves, NdWFamide modulated the motile activities of the tissues. Thus, NdWFamide seems to be a versatile neurotransmitter/modulator of Aplysia and probably regulates the physiological activities of this animal.

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.

Aplysia cardioactive peptide (NdWFamide) enhances the L-type Ca2+ current of Aplysia ventricular myocytes.

NdWFamide is a D-amino acid containing tripeptide purified from Aplysia heart. Although the cardioexcitatory action of NdWFamide is well established, little is known about how the excitatory action is induced. To examine the action of the peptide on the ion channels expressed in the Aplysia heart muscles, we carried out whole cell clamp experiments in the isolated Aplysia ventricular myocytes. We found that the high voltage-activated (HVA) Ca(2+) current of Aplysia ventricular myocytes is mostly a nifedipine-sensitive L-type current, and that the current was enhanced by NdWFamide via the activation of G proteins.

The enterins inhibit the contractile activity of the anterior aorta of Aplysia kurodai.

The anterior aorta is one of the largest blood vessels in the marine mollusc Aplysia kurodai. We examined the actions of recently identified neuropeptides, the enterins, on this blood vessel. Immunohistochemistry revealed that the enterin-immunopositive nerve fibers and varicosity-like structures are abundant in the aorta. When the enterins were applied to the aorta, the basal tonus of the arterial muscles was diminished. The enterins also decreased the contraction amplitude of the anterior aorta evoked either by the application of an Aplysia cardioactive peptide, NdWFamide, or by the stimulation of a nerve innervating the aorta (the vulvar nerve). We found that the enterins activate the 4-aminopyridine (4-AP)-sensitive K(+) channels, and thereby hyperpolarize the membrane potential of the aortic muscles. In the presence of 4-AP, the enterins failed to inhibit the muscle contraction evoked by the vulvar nerve stimulation, suggesting that the inhibition is mainly due to the activation of the 4-AP-sensitive K(+) channels. The inhibition of the NdWFamide-evoked contraction by the enterin was not, however, affected by 4-AP. These results suggest that the enterins are involved in inhibitory regulation of the contractile activity of the anterior aorta, and that the inhibition could be due to multiple mechanisms.