Osamu Matsushima

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.

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.

Novel excitatory neuropeptides isolated from a prosobranch gastropod, Thais clavigera : The molluscan counterpart of the annelidan GGNG peptides

The GGNG peptides are excitatory neuropeptides identified from earthworms, leeches and polychaeta. Two structurally related peptides were purified and characterized from a mollusk, Thais clavigera (prosobranch gastropod). The peptides designated as Thais excitatory peptide-1 (TEP-1) (KCSGKWAIHACWGGN-NH2) and TEP-2 (KCYGKWAMHACWGGN-NH2) are pentadecapeptides having one disulfide bond and C-terminal GGN-NH2 structures, which are shared by most GGNG peptides. TEP augmented the motilities of Thais esophagus and penial complex. TEP-like immunoreactivity is distributed in both the neurons of the central nervous system and nerve endings in the penial complex. Thus, the involvement of TEP in the contraction of the digestive and reproductive systems is suggested. Substitution of amino acids in TEP revealed that two tryptophan residues in TEP are important for maintaining bioactivity.

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.

The amino-terminal region of hemokinin-1 regulates the induction of thermal hyperalgesia in rats

It is known that intrathecal administration of substance P (SP) induces thermal hyperalgesia, whereas hemokinin-1 (HK-1), a member of the same tachykinin family as SP, hardly induces thermal hyperalgesia; however, the underlying mechanism remains to be elucidated. Therefore, we aimed to clarify which amino acid of these peptides contributes to the induction of thermal hyperalgesia. When two chimera peptides between the N-terminal region of SP and the C-terminal region of HK-1, and vice versa, SP (1-5)/HK-1 and HK-1 (1-5)/SP, were intrathecally administered, SP (1-5)/HK-1 induced thermal hyperalgesia whereas HK-1 (1-5)/SP had hardly any effect; furthermore, thermal hyperalgesia was induced by only C-terminal fragments of HK-1 and SP. These findings indicate that the N-terminal region of HK-1 is involved in the non-induction of thermal hyperalgesia. Next, we synthesized and intrathecally administered these chimera peptides in which part of the N-terminal region of HK-1 was replaced with that of SP, and vice versa, and all synthesized peptides induced thermal hyperalgesia. Both SP (1-2)/HK-1 and HK-1 (1-4)/SP certainly induced thermal hyperalgesia, although HK-1 and HK-1 (1-5)/SP had hardly any effect; therefore, it is probable that Ser at the 2nd position and Arg at the 5th position of HK-1 may be involved in the non-induction of thermal hyperalgesia. Furthermore, peptides in which amino acid at the 3rd and/or 4th positions of HK-1 was replaced with that of SP were synthesized. Intrathecal administration of HK-1 (1-2,4-5)/SP, but not HK-1 (1-2,5)/SP and HK-1 (1-3,5)/SP, hardly induced thermal hyperalgesia. These findings indicate that three amino acids, Ser, Thr and Arg at the 2nd, 4th and 5th positions of HK-1, respectively, regulate the induction of thermal hyperalgesia by HK-1.

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.

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.

An amino-terminal fragment of hemokinin-1 has an inhibitory effect on pruritic processing in rats.

Hemokinin-1 (HK-1) is a peptide encoded by the preprotachykinin gene, TAC-4, and shares the hydrophobic carboxyl-terminal (C-terminal) region common to mammalian tachykinin peptides, such as substance P (SP). It is generally believed that C-terminal fragments of SP elicit an excitatory effect, while pretreatment with amino-terminal (N-terminal) fragments of SP inhibits the function of SP; however, there is no available information on HK-1. Therefore, to clarify the characteristics of C-terminal and N-terminal fragments of HK-1, HK-1 was divided into HK-1 (1-5) as the N-terminal fragment and HK-1 (6-11) as the C-terminal fragment based on the similarity of amino acids between HK-1 and SP. Intrathecal administration of HK-1 (6-11) induced scratching behavior similar to HK-1, while HK-1 (1-5) hardly induced scratching. Pretreatment with HK-1 (1-5), however, attenuated scratching induced by HK-1 and SP, whereas pretreatment with SP (1-5) attenuated SP-induced scratching, but not HK-1. Furthermore, intrathecal administration of HK-1 (1-5) and SP (1-5) markedly attenuated the induction of flinching and enhancement of c-Fos expression in the spinal cord following the intradermal administration of formalin, a noxious stimulant, while pretreatment with HK-1 (1-5), but not SP (1-5), markedly attenuated the induction of scratching behavior by subcutaneous administration of pruritic agents, such as serotonin or histamine. Taken together, these findings indicate that HK-1 (1-5) suppresses pruritic and nociceptive processing, while SP (1-5) suppresses nociceptive processing. Therefore, it is suggested that HK-1 (1-5) may be a useful tool for revealing pruritic processing and HK-1 may play a crucial role in pruritic processing.

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.

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.