help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2003-0359
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ukena, K.
Right arrow Articles by Tsutsui, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ukena, K.
Right arrow Articles by Tsutsui, K.
Endocrinology Vol. 144, No. 9 3879-3884
Copyright © 2003 by The Endocrine Society

Novel Neuropeptides Related to Frog Growth Hormone-Releasing Peptide: Isolation, Sequence, and Functional Analysis

Kazuyoshi Ukena, Aya Koda, Kazutoshi Yamamoto, Tetsuya Kobayashi, Eiko Iwakoshi-Ukena, Hiroyuki Minakata, Sakae Kikuyama and Kazuyoshi Tsutsui

Laboratory of Brain Science, Faculty of Integrated Arts and Sciences, Hiroshima University (K.U., K.T.), Higashi-Hiroshima 739-8521, Japan; Core Research for Evolutional Science and Technology, Japan Science and Technology Corp. (K.U., K.T.), Tokyo 150-0002, Japan; Department of Biology, Waseda University School of Education (A.K., K.Y., S.K.), Nishiwaseda, Tokyo 169-8050, Japan; Department of Regulation Biology, Faculty of Science, Saitama University (T.K.), Shimo-Okubo, Saitama 338-8570, Japan; and Suntory Institute for Bioorganic Research (E.I.-U., H.M.), Mishima, Osaka 618-8503, Japan

Address all correspondence and requests for reprints to: Kazuyoshi Tsutsui, Ph.D., Laboratory of Brain Science, Faculty of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521, Japan. E-mail: tsutsui{at}hiroshima-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We previously identified in the bullfrog a novel hypothalamic RFamide peptide (SLKPAANLPLRF-NH2) that stimulated GH release in vitro and in vivo and therefore was designated frog GH-releasing peptide (fGRP). Molecular cloning of cDNA encoding the deduced fGRP precursor polypeptide further revealed that it encodes fGRP and its related peptides (fGRP-RP-1, -RP-2, and -RP-3). In this study immunoaffinity purification using the antibody against fGRP was therefore conducted to determine whether these three putative fGRP-RPs exist as mature endogenous ligands in the frog brain. The mass peaks of the isolated immunoreactive substances were detected at 535.78, 1034.14, and 1079.71 m/z ([M+2H]2+), and their sequences, SIPNLPQRF-NH2, YLSGKTKVQSMANLPQRF-NH2, and AQYTNHFVHSLDTLPLRF-NH2, were revealed by the fragmentation, showing mature forms encoded in the cDNA sequences of fGRP-RP-1, -RP-2, and -RP-3, respectively. All of these fGRP-RPs contained a C-terminal -LPXRF-NH2 (X = L or Q) sequence, such as fGRP. This study further analyzed hypophysiotropic activities of the identified endogenous fGRP-RPs. Only fGRP-RP-2 stimulated, in a dose-related way, the release of PRL from cultured frog pituitary cells; its threshold concentration ranged from less than 10-7 M. A similar stimulatory action of fGRP-RP-2 on GH release was evident. It was ascertained that fGRP-RP-2 was also effective in elevating the circulating GH and PRL levels when administered systemically. In contrast, fGRP-RPs did not have any appreciable effect on the release of gonadotropins. Thus, fGRP-RP-2 may act as a novel hypothalamic factor on the frog pituitary to stimulate the release of GH and PRL.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
STIMULATED by the discovery of the molluscan neuropeptide Phe-Met-Arg-Phe-NH2 (FMRFamide) existing in the ganglia of the Venus clam (1), neuropeptides with the RFamide motif at their C-termini (RFamide peptides) have been identified in the brains of vertebrate species (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12). In the bullfrog, we previously identified Ser-Leu-Lys-Pro-Ala-Ala-Asn-Leu-Pro-Leu-Arg-Phe-NH2, a novel hypothalamic RFamide peptide (13). The cell bodies and terminals containing this peptide were localized in the suprachiasmatic nucleus and median eminence, respectively (13). This peptide was further revealed to have a considerable GH-releasing activity in vitro and in vivo and hence was designated frog GH-releasing peptide (fGRP) (13). The peptide with the same amino acid sequence as that of fGRP has also been isolated from the brain of European green frog and localized in the hypothalamus and spinal cord (14).

In amphibians, GH secretion from the somatotrophs is known to be under dual control: stimulatory and inhibitory. GHRH (for a review, see Ref.15), pituitary adenylate cyclase-activating polypeptide (15, 16), and ghrelin (17) of amphibian origin have been nominated as stimulating factors. In contrast, frog somatostatin (18) has been described as an inhibiting factor. In addition to these findings, the recent discovery of fGRP, a novel amphibian hypothalamic neuropeptide, has provided the potential for new insight into the regulation of GH secretion.

Recently, molecular cloning of cDNA encoding the precursor of fGRP was attempted in the bullfrog brain (19). Interestingly, analysis of the resulting cDNA revealed that the deduced fGRP precursor encoded one fGRP and three putative gene-related peptide sequences (fGRP-RP-1, -RP-2, and -RP-3) that were invariably equipped with -LPXRF (X = L or Q) at their C termini (19). The present study was conducted to determine whether these three putative peptides exist as translation products in the frog brain and whether they have any hypophysiotropic activity. Here we report that the identified gene-related peptide fGRP-RP-2 may act as a novel mature peptide on the frog anterior pituitary, stimulating the release of both GH and PRL.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Affinity purification and immunoassay
To identify endogenous fGRP-related peptides (fGRP-RPs) in the bullfrog brain, we employed immunoaffinity purification using the specific antibody against fGRP (13). The antiserum was raised in the rabbit, and its specificity has been described previously (13).

The brains of 50 adult male bullfrogs were dissected out, frozen immediately in liquid nitrogen, and kept at -80 C until used. The tissues were boiled and homogenized in 5% acetic acid as described previously (7, 13). The homogenate was centrifuged at 15,000 x g for 20 min at 4 C, and the supernatant was collected. After precipitation with 75% acetone, the supernatant was passed through a disposable C18 cartridge column (Mega Bond-Elut; Varian, Harbor City, CA), and the retained material was loaded onto an immunoaffinity column. The affinity chromatography was carried out as described previously (11, 12). The antibodies against fGRP were conjugated to cyanogen bromide-activated Sepharose 4B as an affinity ligand. The brain extract was applied to the column at 4 C, and the adsorbed materials were eluted with 0.3 M acetic acid containing 0.1% 2-mercaptoethanol. An aliquot of each fraction (1 ml) was analyzed by a dot immunoblot assay with the antibody against fGRP according to our previous method (13). The immunoreactive fractions were concentrated and subjected to a reverse phase HPLC column (ODS-80TM, Tosoh, Tokyo, Japan) with a linear gradient of 20–40% acetonitrile containing 0.1% trifluoroacetic acid for 100 min at a flow rate of 0.5 ml/min. The isolated immunoreactive substances were then subjected to mass spectrometric analyses as described below.

Mass spectrometry and peptide synthesis
After evaporation of the immunoreactive material, the residue was dissolved in 50% methanol containing 0.1% formic acid, and the molecular mass was analyzed by nanoflow electrospray ionization time of flight mass spectrometry (nano ESI-TOF-MS; Q-TOF, Micromass UK, Wythenshawe, UK) as described previously (8, 11, 12). The prospected mass value of each deduced fGRP-RP was calculated using the ProteinProspector program (University of California, San Francisco, CA), and a corresponding peak was further examined in a tandem MS analysis. The needle voltage was optimized at 1000 V, and the cone voltage was set at 50 V. Argon was used as the collision gas, and the energy was set at 50 V.

The peptides with the suggested sequences (fGRP-RP-1, -RP-2, and -RP-3) were synthesized by a manual method, followed by a hydrogen fluoride-anisole cleavage and were purified by reverse phase HPLC. The synthetic peptides were compared with the native ones with regard to behavior on HPLC and mass spectrometry.

In vitro assay
Dispersed anterior pituitary cells of adult male bullfrogs were prepared as previously described (20). The completely dispersed cells were then resuspended in 67% medium 199 (Nissui Pharmaceutical, Tokyo, Japan) containing 0.1% BSA. An aliquot of the cell suspension was used to count the cell number. The volume of the cell suspension was adjusted so that 1 ml contained 3 x 105 cells. Sixty thousand cells in 200 µl medium were plated in each well of a 96-multiwell plate (Corning, Inc., Corning, NY) and preincubated for 24 h at 23 C in an atmosphere of 95% air/5% CO2. Preincubated pituitary cells were transferred to the medium containing various concentrations of the identified fGRP-RP-1, -RP-2, and -RP-3 as mature endogenous ligands and incubated for 24 h at 23 C unless otherwise stated. Anterior pituitary cells were incubated in medium 199 with no additive as controls. After incubation, each medium was centrifuged, and the supernatant was subjected to RIAs for bullfrog GH (21), PRL (22), LH (23), and FSH (24).

In vivo assay
Juvenile frogs, weighing 12–15 g, were injected ip with 40 µg fGRP-RP-2, a mature endogenous ligand, in 100 µl saline or saline only. They were killed 0, 1, 2, 6, and 12 h after the injection, and blood samples were collected as described previously (13). Plasma GH and PRL concentrations were determined by RIAs for bullfrog GH (21) and PRL (22), respectively.

RIA
Bullfrog GH (25) and PRL (26) used as standards and radioligands were previously purified in our laboratory. Purified bullfrog LH and FSH (27) were provided by Dr. S. Tanaka (Shizuoka University, Shizuoka, Japan). They were radioiodinated with 125I (Na125I, Radiochemical Center, Amersham Pharmacia Biotech, Little Chalfont, UK) by the method described previously (21, 22, 23, 24). Intraassay coefficients of variation in the RIA for GH, PRL, LH, and FSH were 4.1%, 3.8%, 3.6%, and 4.3%, respectively; and interassay coefficients of variation were 4.3%, 3.3%, 3.6%, and 3.9%, respectively. The RIA results of bullfrog GH, PRL, LH, and FSH assayed in duplicate were calculated in terms of nanograms per 104 cells per 24 h.

Statistical analysis
Results of the RIAs were expressed as the mean ± SEM. The effects of mature endogenous fGRP-RPs on the release of GH, PRL, LH, and FSH from the bullfrog pituitaries were analyzed for significance by one-way ANOVA. If significant by ANOVA, these analyses were followed by Duncan’s multiple range test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of mature endogenous fGRP-RPs
Acetic acid extracts of bullfrog brains were passed through a disposable C18 reverse phase cartridge column. The retained material eluted with 60% methanol was then subjected to affinity chromatography with the antibody against fGRP. Immunoreactivity was measured in the eluted fractions by a dot immunoblot assay. Immunoreactive fractions were subjected to the reverse phase HPLC purification, and the eluate was fractionated every 2 min and assayed by immunoblotting. The fractions corresponding to the elution times of 20–22, 22–24, and 56–58 min showed intense immunoreactivities (Table 1Go). Each purified substance was further examined by mass spectrometry. The mass values of predicted peptides were previously calculated using programming software on the basis of the sequence of fGRP preproprotein (19). A molecular ion peak in the spectrum of each substance was observed at 535.78, 1034.14, or 1079.71 m/z ([M+2H]2+) on the nano ESI-TOF-MS (Table 1Go). These values were close to the mass numbers of 535.81, 1034.06, and 1079.56 m/z ([M+2H]2+) calculated for three deduced fGRP-RP-1, -RP-2, and -RP-3, respectively (Table 1Go). Therefore, the sequence of each substance was determined by a tandem MS analysis (Fig. 1Go). Figure 1AGo shows the fragmentation patterns of fGRP-RP-2. Assignment of the observed typical fragment ions, i.e. N-terminal (b) and C-terminal (y) ions, indicated that the amino acid sequence of this peak was compatible with the sequence of fGRP-RP-2, YLSGKTKVQSMANLPQRF-NH2 (Figs. 1BGo and 2BGo). Similar analyses of fGRP-RP-1 and -RP-3 were performed, and the sequences were shown as follows: SIPNLPQRF-NH2 (fGRP-RP-1; Fig. 2BGo) and AQYTNHFVHSLDTLPLRF-NH2 (fGRP-RP-3; Fig. 2BGo). To confirm the data obtained by these structural analyses, the peptides with the suggested sequences were synthesized and compared with the purified peptides with regard to retention time on HPLC and mass number. Both native and synthetic peptides showed a similar retention time on the reverse phase HPLC and a similar molecular mass in all three fGRP-RPs (Table 1Go). Furthermore, the fragmentation of each synthetic fGRP-RP by the tandem MS analysis was completely coincident with that of the native one (data not shown).


View this table:
[in this window]
[in a new window]
  		TABLE 1. Behavior of native and synthetic peptides on HPLC and MS

 


View larger version (29K):
[in this window]
[in a new window]
  		FIG. 1. Fragmentation patterns of the purified substance with the observed mass number of 1034.14 m/z ([M+2H]2+) by a tandem MS analysis (A). The spectrum shows typical mass values of predicted fGRP-RP-2 fragment ions. The observed N-terminal (b) and C-terminal (y) fragmentation ions are assigned in the sequence of fGRP-RP-2 (B).

 


View larger version (38K):
[in this window]
[in a new window]
  		FIG. 2. Amino acid sequence of the fGRP preproprotein (A) (19 ). fGRP (13 ) and fGRP-RPs identified in this study are boxed. Amino acid sequences of the identified mature peptides, fGRP and fGRP-RPs (B). All of the identified fGRP-RPs contain a C-terminal -LPXRF-NH2 (X = L or Q) sequence.

 
In vitro effects of fGRP-RPs on the release of GH, PRL, and gonadotropins from pituitary cells
Using primary cultures of bullfrog anterior pituitary cells, we conducted experiments to ascertain whether the mature endogenous fGRP-RPs have any influence on the release of pituitary hormones such as GH, PRL, LH, and FSH. Firstly, we studied the effects of the three fGRP-RPs at various concentrations (10-7-10-5 M) on the release of GH, PRL, LH, and FSH during a 24-h incubation. As shown in Fig. 3Go, A and B, fGRP-RP-2 stimulated the release of both GH and PRL, whereas fGRP-RP-1 and -RP-3 had no activity in this respect. On the other hand, none of the three fGRP-RPs had a significant effect on the release of LH and FSH (Fig. 3Go, C and D). As shown in Fig. 3Go, A and B, the stimulatory effects of fGRP-RP-2 on the release of GH and PRL were dose dependent. The threshold concentrations on the release of GH and PRL ranged between 10-7 and 10-6 M and less than 10-7 M, respectively (Fig. 3Go, A and B). The stimulatory effect of fGRP-RP-2 at 10-5 M on GH release was almost the same as that of fGRP at the same dose (Fig. 3AGo). In contrast to fGRP-RP-2, fGRP did not show any significant effect on PRL release (Fig. 3B).



View larger version (25K):
[in this window]
[in a new window]
  		FIG. 3. Effects of fGRP-RP-1, -RP-2, and -RP-3 on the release of GH (A), PRL (B), LH (C), and FSH (D) from cultured bullfrog pituitary cells after a 24-h incubation at 23 C with different concentrations (10-5-10-7 M) of the peptides. Each column and vertical line represent the mean ± SEM of determinations from six different wells. **, P < 0.01; *, P < 0.05 (vs. control group).

 
Subsequently, we studied the effects of fGRP-RP-2 on the release of GH and PRL (10-5 M) at varying incubation times (12, 24, and 36 h). As shown in Fig. 4Go, A and B, the release of both GH and PRL increased according to the length of incubation time with fGRP-RP-2. In both cases, the stimulatory effect of fGRP-RP-2 became evident by the first 12 h of incubation (Fig. 4Go, A and B). In the case of GH, the release reached a plateau at a 24-h incubation (Fig. 4AGo), whereas PRL was released continuously during a 36-h incubation (Fig. 4BGo).



View larger version (14K):
[in this window]
[in a new window]
  		FIG. 4. Release of GH (A), and PRL (B) from cultured bullfrog pituitary cells during different incubation times with ({bullet}) or without ({circ}) fGRP-RP-2 (10-5 M) at 23 C. Each circle and vertical line represent mean ± SEM of determinations from six different wells. **, P < 0.01; *, P < 0.05 (vs. control group).

 
In vivo effects of fGRP-RP-2 on circulating GH and PRL levels
The effects of an ip injection of fGRP-RP-2 on circulating GH and PRL levels became conspicuous (P < 0.01 or 0.05) within 2 h (Fig. 5Go, A and B). The hormone levels declined thereafter. As in the case of the in vitro experiment, the effect of fGRP-RP-2 on GH release (Fig. 5AGo) was more pronounced than that on PRL release (Fig. 5BGo).



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 5. Effect of fGRP-RP-2 on the circulating levels of GH (A) and PRL (B). Juvenile frogs received an ip injection of 40 µg fGRP-RP-2 in 100 µl saline ({bullet}) or 100 µl saline alone ({circ}). Each circle and vertical line represent the mean ± SEM of determinations from six individual animals. **, P < 0.01; *, P < 0.05 (vs. control group).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We identified three fGRP-RPs as mature endogenous ligands using immunoaffinity purification. On the basis of the results of structure determinations, such as molecular weight presumption, analysis of fragmentation patterns, and comparison of HPLC behavior, the present study indicated that the bullfrog brain expresses not only fGRP (13) but also three fGRP-RPs as mature endogenous peptides equipped with -LPXRF-NH2 (X = L or Q) at their C termini. Their sequences of identified fGRP-RP-1, -RP-2, and -RP-3 are flanked on both ends by the typical endoproteolytic sequences, i.e. KIAK or RFGR. Therefore, some subtilisin/kexin-like proprotein convertases may be involved in the cleavage of the precursor polypeptide (for a review, see Ref.28). Indeed, the proprotein convertases PC1, PC2, and PC5-A have been identified in the frog brain, including the hypothalamus (29, 30, 31).

Previously we demonstrated that fGRP selectively stimulates the release of GH from the anterior pituitary of the bullfrog both in vivo and in vitro (13). In the present study we further characterized the hypophysiotropic activities of the identified fGRP-RPs. Among the three fGRP-RPs, fGRP-RP-2 was shown to have a GH-releasing activity. It should be mentioned that the sequences of two amino acids (Ala and Asn) before the LPXRFamide motif in fGRP-RP-2, but not other two fGRP-RPs, are identical with those in fGRP. Unlike fGRP, fGRP-RP-2 was further revealed to have a moderate PRL-releasing activity. fGRP-RP-2 stimulated, in a dose-related manner, PRL release from cultured frog pituitary cells. Such a stimulatory effect may be taken as a physiological action, because its threshold concentration ranged from less than 10-7 M. We also confirmed that exogenously administered fGRP-RP-2 elevated the PRL level in the circulating blood of juvenile frogs. In contrast to fGRP-RP-2, two other fGRP-RPs did not show any appreciable effect on the release of PRL. Our previous results of in situ hybridization revealed that the mRNA encoding fGRP precursor polypeptide was only expressed in the suprachiasmatic nucleus of the bullfrog hypothalamus (19). In addition, in our previous immunocytochemical analysis using the antiserum that cross-reacts with fGRP and fGRP-RPs (13), immunoreactive cell bodies and terminals were localized in the suprachiasmatic nucleus and median eminence, respectively. Taken together, these results suggest that fGRP and fGRP-RP-2 are synthesized by the same neurons and act directly on the frog anterior pituitary through the median eminence to stimulate the release of GH and PRL (only fGRP-RP-2). However, more precise experiments using the distinguishable antibodies between fGRP and fGRP-RP-2 are needed to address the site of production.

The simultaneous stimulation of fGRP-RP-2 on the release of GH and PRL is not unprecedented. Other hypophysiotropic peptides in amphibians are known to release two hormones simultaneously. Bullfrog ghrelin that was recently isolated (17) as well as TRH (32, 33) have a potency to stimulate both GH and PRL from the bullfrog pituitary. It was noted that there is some difference in the responsiveness to fGRP-RP-2 between GH and PRL cells. A high concentration (10-5 M) of fGRP-RP-2 strongly stimulated the release of GH compared with that of PRL, whereas the stimulatory activity on PRL cells, but not GH cells, was still retained when the concentration was reduced to 10-7 M. This may reflect the difference in physicochemical properties of the receptor for this peptide between GH and PRL cells. It was confirmed that when the concentration of fGRP-RP-2 was reduced to 10-8 M, it did not show PRL-releasing activity (data not shown). When fGRP-RP-2 was administered ip, the response of both GH and PRL cells was acute and pronounced compared with the response observed in the static incubation. This may be attributable to the difference between intact and dispersed pituitary cells. If the pituitary is supplied with blood circulation, and the cells are closely in contact with other neighboring cells, which may release paracrine factors (32, 34), their responsiveness to a certain secretagogue will be enhanced. Attenuation of the effect of a single injection of fGRP-RP-2 on the release of both GH and PRL by 6 h may be the result of degradation of the peptide in the circulation.

Recently, neuropeptides possessing the C-terminal LPXRFamide motif (LPXRFamide peptide) have been found in the brains of mammals (9, 10, 11), birds (7, 8), amphibians (13, 14, 19), and fishes (12). In this study fGRP and fGRP-RPs, frog LPXRFamide peptides, did not induce any significant effect on the release of gonadotropins. However, the quail LPXRFamide peptide (SIKPSAYLPLRF-NH2), which has a high sequence homology (75%) with fGRP (SLKPAANLPLRF-NH2), exerted an inhibitory effect on the release of gonadotropins from the cultured quail anterior pituitary (7). We therefore termed it gonadotropin-inhibitory hormone (GnIH) (7). Molecular cloning of cDNA encoding GnIH from the quail brain revealed that it encoded GnIH and its related peptides (GnIH-RP-1 and -RP-2), which also contained a C-terminal LPXRFamide motif (8). The possible hypophysiotropic functions of GnIH-RPs are still uncertain in the bird. On the other hand, the mammalian LPXRFamide peptides [RFRP-1, SLTFEEVKDWAPKIKMNKPVVNKMPPSAANLPLRF-NH2 (10); RFRP-2, ANMEAGTMSHFPSLPQRF-NH2 (11)] may have PRL-releasing activity (9). Taken together, these findings strengthen the view that there may be a group of LPXRFamide peptides contributing to the multifactorial regulation of pituitary hormone release in vertebrates.


   Acknowledgments
 
We thank Dr. George Bentley (University of Washington, Seattle, WA) for his valuable discussion and for reading the manuscript.


   Footnotes
 
This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan [12440233, 12894021, 13210101, 15207007 (to K.T.), 12440234 (to S.K.), and 15770040 (to K.U.)]; Waseda University Grant for Special Research Projects, Tokyo, Japan (to S.K.); and the Sunbor Grant from Suntory Institute for Bioorganic Research, Osaka, Japan (to K.U.).

K.U. and A.K. contributed equally to this work.

Abbreviations: fGRP, Frog GH-releasing peptide; fGRP-RP, frog GH-releasing peptide-related peptide; GnIH, gonadotropin-inhibitory hormone.

Received March 24, 2003.

Accepted for publication May 19, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Price DA, Greenberg MJ 1977 Structure of a molluscan cardioexcitatory neuropeptide. Science 197:670–671[Abstract/Free Full Text]
  2. Dockray GJ, Reeve Jr JR, Shively J, Gayton RJ, Barnard CS 1983 A novel active pentapeptide from chicken brain identified by antibodies to FMRFamide. Nature 305:328–330[CrossRef][Medline]
  3. Yang HY, Fratta W, Majane EA, Costa E 1985 Isolation, sequencing, synthesis, and pharmacological characterization of two brain neuropeptides that modulate the action of morphine. Proc Natl Acad Sci USA 82:7757–7761[Abstract/Free Full Text]
  4. Perry SJ, Yi-Kung Huang E, Cronk D, Bagust J, Sharma R, Walker RJ, Wilson S, Burke JF 1997 A human gene encoding morphine modulating peptides related to NPFF and FMRFamide. FEBS Lett 409:426–430[CrossRef][Medline]
  5. Hinuma S, Habata Y, Fujii R, Kawamata Y, Hosoya M, Fukusumi S, Kitada C, Masuo Y, Asano T, Matsumoto H, Sekiguchi M, Kurokawa T, Nishimura O, Onda H, Fujino M 1998 A prolactin-releasing peptide in the brain. Nature 393:272–276[CrossRef][Medline]
  6. Fujimoto M, Takeshita K, Wang X, Takabatake I, Fujisawa Y, Teranishi H, Ohtani M, Muneoka Y, Ohta S 1998 Isolation and characterization of a novel bioactive peptide, Carassius RFamide (C-RFa), from the brain of the Japanese crucian carp. Biochem Biophys Res Commun 242:436–440[CrossRef][Medline]
  7. Tsutsui K, Saigoh E, Ukena K, Teranishi H, Fujisawa Y, Kikuchi M, Ishii S, Sharp PJ 2000 A novel avian hypothalamic peptide inhibiting gonadotropin release. Biochem Biophys Res Commun 275:661–667[CrossRef][Medline]
  8. Satake H, Hisada M, Kawada T, Minakata H, Ukena K, Tsutsui K 2001 Characterization of a cDNA encoding a novel avian hypothalamic neuropeptide exerting an inhibitory effect on gonadotropin release. Biochem J 354:379–385[CrossRef][Medline]
  9. Hinuma S, Shintani Y, Fukusumi S, Iijima N, Matsumoto Y, Hosoya M, Fujii R, Watanabe T, Kikuchi K, Terao Y, Yano T, Yamamoto T, Kawamata Y, Habata Y, Asada M, Kitada C, Kurokawa T, Onda H, Nishimura O, Tanaka M, Ibata Y, Fujino M 2000 New neuropeptides containing carboxy-terminal RFamide and their receptor in mammals. Nat Cell Biol 2:703–708[CrossRef][Medline]
  10. Fukusumi S, Habata Y, Yoshida H, Iijima N, Kawamata Y, Hosoya M, Fujii R, Hinuma S, Kitada C, Shintani Y, Suenaga M, Onda H, Nishimura O, Tanaka M, Ibata Y, Fujino M 2001 Characteristics and distribution of endogenous RFamide-related peptide-1. Biochim Biophys Acta 1540:221–232[Medline]
  11. Ukena K, Iwakoshi E, Minakata H, Tsutsui K 2002 A novel rat hypothalamic RFamide-related peptide identified by immunoaffinity chromatography and mass spectrometry. FEBS Lett 512:255–258[CrossRef][Medline]
  12. Sawada K, Ukena K, Satake H, Iwakoshi E, Minakata H, Tsutsui K 2002 Novel fish hypothalamic neuropeptide: cloning of a cDNA encoding the precursor polypeptide and identification and localization of the mature peptide. Eur J Biochem 269:6000–6008[Medline]
  13. Koda A, Ukena K, Teranishi H, Ohta S, Yamamoto K, Kikuyama S, Tsutsui K 2002 A novel amphibian hypothalamic neuropeptide: isolation, localization and biological activity. Endocrinology 143:411–419[Abstract/Free Full Text]
  14. Chartrel N, Dujardin C, Leprince J, Desrues L, Tonon MC, Cellier E, Cosette P, Jouenne T, Simonnet G, Vaudry H 2002 Isolation, characterization, and distribution of a novel neuropeptide, Rana RFamide (R-RFa), in the brain of the European green frog Rana esculenta. J Comp Neurol 448:111–127[CrossRef][Medline]
  15. Montero M, Yon L, Kikuyama S, Dufour S, Vaudry H 2000 Molecular evolution of the growth hormone-releasing hormone/pituitary adenylate cyclase activating polypeptide gene family. Functional implication in the regulation of growth hormone secretion. J Mol Endocrinol 25:157–168[Abstract]
  16. Martinez-Fuentes AJ, Gonzalez de Aguilar JL, Lacuisse S, Kikuyama S, Vaudry H, Gracia-Navarro F 1994 Effect of frog pituitary adenylate cyclase activating polypeptide (PACAP) on amphibian pituitary cells. In: Rosselin G, ed. Vasoactive intestinal peptide, pituitary adenylate cyclase activating polypeptide and related regulatory peptides: from molecular biology to clinical applications. Singapore: World Scientific; 376–380
  17. Kaiya H, Kojima M, Hosoda H, Koda A, Yamamoto K, Kitajima Y, Matsumoto M, Minamitake Y, Kikuyama S, Kangawa K 2001 Bullfrog ghrelin is modified by n-octanoic acid at its third threonine residue. J Biol Chem 276:40441–40448[Abstract/Free Full Text]
  18. Jeandel L, Okuno A, Kobayashi T, Kikuyama S, Tostivint H, Lihrmann I, Chartrel N, Conlon JM, Fournier A, Tonon MC, Vaudry H 1998 Effects of the two somatostatin variants somatostatin-14 and [Pro2,Met13]somatostatin-14 on receptor binding, adenylyl cyclase activity and growth hormone release from the frog pituitary. J Neuroendocrinol 10:187–192[CrossRef][Medline]
  19. Sawada K, Ukena K, Kikuyama S, Tsutsui K 2002 Identification of a cDNA encoding a novel amphibian growth hormone-releasing peptide and localization of its transcript. J Endocrinol 174:395–402[Abstract]
  20. Oguchi A, Tanaka S, Yamamoto K, Kikuyama S 1996 Release of {alpha}-subunit of glycoprotein hormones from the bullfrog pituitary: possible effect of {alpha}-subunit on prolactin cell function. Gen Comp Endocrinol 102:141–146[CrossRef][Medline]
  21. Kobayashi T, Kikuyama S 1991 Homologous radioimmunoassay for bullfrog growth hormone. Gen Comp Endocrionol 82:14–22
  22. Yamamoto K, Kikuyama S 1982 Radioimmunoassay of prolactin in plasma of bullfrog tadpoles. Endocrinol Jpn 29:159–167[Medline]
  23. Tanaka S, Hanaoka Y, Wakabayashi K 1983 A homologous radioimmunoassay for bullfrog basic gonadotropin. Endocrinol Jpn 29:159–167
  24. Polzonetti-Magni AM, Mosconi G, Carnevali O, Yamamoto K, Hanaoka Y, Kikuyama S 1998 Gonadotropins and reproductive function in the anuran amphibian, Rana esculenta. Biol Reprod 58:88–93[Abstract/Free Full Text]
  25. Kobayashi T, Kikuyama S, Yasuda A, Kawauchi H, Yamaguchi K, Yokoo Y 1989 Purification and characterization of bullfrog growth hormone. Gen Comp Endocrinol 73:417–424[CrossRef][Medline]
  26. Yamamoto K, Kikuyama S 1981 Purification and properties of bullfrog prolactin. Endocrinol Jpn 28:59–64[Medline]
  27. Takahashi H, Hanaoka Y 1981 Isolation and characterization of multiple components of basic gonadotropin from bullfrog (Rana catesbeiana) pituitary gland. J Biochem 90:1333–1340[Abstract/Free Full Text]
  28. Seidah NG, Chrétien M 1999 Proprotein and prohormone convertases: a family of subtilases generating diverse bioactive polypeptides. Brain Res 848:45–62[CrossRef][Medline]
  29. Vieau D, Gangnon F, Jégou S, Danger JM, Vaudry H 1998 Characterization of the cDNA encoding the prohormone convertase PC2 and localization of the mRNA in the brain of the frog Rana ridibunda. Mol Brain Res 63:1–13[Medline]
  30. Gangnon F, Danger JM, Jegou S, Vieau D, Seidah NG, Vaudry H 1999 Molecular cloning, characterization of cDNA, and distribution of mRNA encoding the frog prohormone convertase PC1. J Comp Neurol 405:160–172[CrossRef][Medline]
  31. Gangnon F, Jégou S, Vallarino M, Vieau D, Vaudry H 2003 Molecular characterization of the cDNA and localization of the mRNA encoding the prohormone convertase PC5-A in the European green frog. J Comp Neurol 456:60–72[CrossRef][Medline]
  32. Aida T, Yamamoto K, Kikuyama S 1999 Enhancement by proopiomelanocortin-derived peptides of growth hormone and prolactin secretion by bullfrog pituitary cells. Gen Comp Endocrinol 115:101–109[CrossRef][Medline]
  33. Koda A, Yamamoto K, Uchiyama H, Vaudry H, Kikuyama S 2000 Effect of activin A and follistatin on the release of pituitary hormones in the bullfrog Rana catesbeiana. Zool Sci 17:971–975[CrossRef]
  34. Maruyama M, Matsumoto H, Fujiwara K, Noguchi J, Kitada C, Fujino M, Inoue K 2001 Prolactin releasing peptide as a novel stress mediator in the central nervous system. Endocrinlogy 142:2032–2038



This article has been cited by other articles:


Home page
 EndocrinologyHome page
Y. R. Lee, K. Tsunekawa, M. J. Moon, H. N. Um, J.-I. Hwang, T. Osugi, N. Otaki, Y. Sunakawa, K. Kim, H. Vaudry, et al.
Molecular Evolution of Multiple Forms of Kisspeptins and GPR54 Receptors in Vertebrates
Endocrinology, June 1, 2009; 150(6): 2837 - 2846.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
V. S. Chowdhury, K. Yamamoto, I. Saeki, I. Hasunuma, T. Shimura, and K. Tsutsui
Melatonin Stimulates the Release of Growth Hormone and Prolactin by a Possible Induction of the Expression of Frog Growth Hormone-Releasing Peptide and Its Related Peptide-2 in the Amphibian Hypothalamus
Endocrinology, March 1, 2008; 149(3): 962 - 970.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
T. Ubuka, S. Kim, Y.-c. Huang, J. Reid, J. Jiang, T. Osugi, V. S. Chowdhury, K. Tsutsui, and G. E. Bentley
Gonadotropin-Inhibitory Hormone Neurons Interact Directly with Gonadotropin-Releasing Hormone-I and -II Neurons in European Starling Brain
Endocrinology, January 1, 2008; 149(1): 268 - 278.
[Abstract] [Full Text] [PDF]

Home page
 J EndocrinolHome page
M. Amano, S. Moriyama, M. Iigo, S. Kitamura, N. Amiya, K. Yamamori, K. Ukena, and K. Tsutsui
Novel fish hypothalamic neuropeptides stimulate the release of gonadotrophins and growth hormone from the pituitary of sockeye salmon.
J. Endocrinol., March 1, 2006; 188(3): 417 - 423.
[Abstract] [Full Text] [PDF]

Home page
 J EndocrinolHome page
H Yin, K Ukena, T Ubuka, and K Tsutsui
A novel G protein-coupled receptor for gonadotropin-inhibitory hormone in the Japanese quail (Coturnix japonica): identification, expression and binding activity
J. Endocrinol., January 1, 2005; 184(1): 257 - 266.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ukena, K.
Right arrow Articles by Tsutsui, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ukena, K.
Right arrow Articles by Tsutsui, K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals