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Department of Biochemistry (H.K., M.K., H.H., K.K.), National Cardiovascular Center Research Institute, Osaka 565-8565, Japan; Laboratory of Comparative Endocrinology (S.V.D.G., S.G., V.M.D.), Zoological Institute, Catholic University of Leuven, B-3000, Leuven, Belgium; Suntory Institute for Medicinal Research and Development (Y.K., M.M.), Gunma 370-0503, Japan; and Division of Molecular Genetics, Institute of Life Science, Kurume University (M.K.), Fukuoka 839-0861, Japan
Address all correspondence and requests for reprints to: Hiroyuki Kaiya, Ph.D., Department of Biochemistry National Cardiovascular Center Research Institute 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan. E-mail: kaiya{at}ri.ncvc.go.jp.
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Introduction |
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Although little is known about ghrelin in nonmammalian vertebrates, we recently identified ghrelin in the stomach of a bullfrog (Rana catesbeiana) and demonstrated that this 28-amino acid peptide was able to stimulate GH- and PRL-release from pituitary cells of the bullfrog itself (27). This finding suggests that ghrelin may exist in a wide variety of lower vertebrates and play a role in pituitary function. Indeed, indirect evidence supporting the presence of ghrelin or a ghrelin-like molecule in chicken has been reported as follows: 1) putative GHS-R homologs have already been partially cloned (GenBank accession numbers AJ309542 and AJ309543); 2) peptidyl and nonpeptidyl GHSs stimulate GH secretion in vivo and in vitro (28, 29, 30); 3) ghrelin-immunoreactive cells are present in several areas of the chicken hypothalamus (31); and 4) human ghrelin increases plasma GH levels in young chickens (31). Furthermore, it has been demonstrated that intracerebroventricular injection of rat ghrelin inhibits food intake in neonatal chicks (32). Taken together, these findings strongly suggest that ghrelin is present in the chicken and that it also plays a regulatory role in GH release and feeding.
In this study, we identified ghrelin and its precursor cDNA from chicken stomach. Chicken ghrelin is a 26-amino acid peptide that contains an n-octanoylated or n-decanoylated serine at position 3. In addition to elevating plasma GH levels in the rat, chicken ghrelin also increased plasma GH and corticosterone levels after iv injection in growing chicks.
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Materials and Methods |
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Chicken proventriculus was collected from a supplier (Koyushokucho Inc., Koyu-gun, Miyazaki, Japan). Frozen tissue (40 g) was pulverized and boiled for 10 min in 5 vol of water to inactivate intrinsic proteases. The sample was then chilled on ice and adjusted to 1 M acetic acid (AcOH) by adding glacial AcOH. The boiled stomach tissue was homogenized using a Polytron mixer (Kinematica, Inc., Lucerne, Switzerland). Crude acid extracts were then centrifuged for 30 min at 10,000 x g. The supernatant was diluted with an equal volume of distilled water and loaded onto a Sep-pak Vac 35-cc cartridge (Waters Corp., Milford, MA) preequilibrated with 0.5 M AcOH. The cartridge was washed with a 10% acetonitrile (ACN)/0.1% trifluoroacetic acid (TFA) solution. Peptides were sequentially eluted with 40 ml of 25% and 60% ACN/0.1% TFA, respectively. A 50-mg tissue equivalent from each fraction was subjected to the FLIPR assay. The active fraction, which eluted at 60% ACN/0.1% TFA, was evaporated and then reconstituted with 1 M AcOH. The resulting extract was subjected to SP-Sephadex C-25 (H+ form) chromatography (Amersham Pharmacia Biotech, Buckinghamshire, UK). Successive elution with 1 M AcOH, 2 M pyridine, and 2 M pyridine-AcOH (pH 5.0), yielded three fractions (SP-I, SP-II, and SP-III, respectively). Half of the SP-III fraction (20-g tissue equivalent), which contained strong basic peptides and ghrelin activity, was dissolved in 10 mM ammonium formate (pH 4.8) containing 10% ACN (solvent A). The sample solution was subjected to carboxymethyl (CM)-ion exchange HPLC at a flow rate of 2 ml/min; this procedure used a TSK-gel CM-2SW column (7.8 x 300 mm; Tosoh, Tokyo, Japan) with a two-step gradient profile, first from solvent A to 25% solvent B (1 M ammonium formate containing 10% ACN, pH 4.8) over 10 min and then to 55% solvent B over 90 min. The eluate was collected in 2-ml fractions over 80 min. The activity of each fraction was determined by subjecting a 50-mg tissue equivalent to FLIPR analysis. Active fractions (groups A–G) were diluted in equal volumes of 0.1% TFA and subjected to Sep-pak Light C18 cartridge (Waters Corp.) purification for removal of excess salts. The sample, eluted with 60% ACN/0.1% TFA, was lyophilized, dissolved in 1 ml of a 100-mM phosphate buffer (pH 7.4), and then subjected to antirat ghrelin [1–11] IgG immunoaffinity purification (27, 33). Adsorbed substances were eluted with 1 ml 60% ACN/0.1% TFA. The eluate was evaporated and then separated by reverse-phase (RP)-HPLC using a µBondasphare C18 column (3.9 x 150 mm, Waters Corp.) at a flow rate of 1 ml/min on a linear gradient from 10% to 60% ACN/0.1% TFA for 40 min. The eluate was collected in 0.5-ml fractions. An aliquot of each fraction (120-mg tissue equivalent) was assayed for ghrelin activity by FLIPR. Active fractions were further purified by RP-HPLC using a diphenyl column, 219TP5215 (2.1 x 150 mm; Vydac, Hesperia, CA) for 80 min on a linear gradient from 10% to 60% ACN/0.1% TFA at a flow rate of 0.2 ml/min. Each absorption peak was collected, and an aliquot of each fraction (200-mg tissue equivalent) was assayed for activity by FLIPR. Some peaks were further purified on a µBondasphare C18 column (2.1 x 150 mm, Waters Corp.) for 80 min. Approximately 10 pmol of the final purified peptide was analyzed by a protein sequencer (model 494; PE Applied Biosystems, Foster City, CA). One picomole was used for molecular weight determination by MALDI-TOF mass spectrometry (Voyager system, PE Applied Biosystems).
3'-RACE (rapid amplification of the cDNA ends)
Total RNA was extracted from 3 g chicken proventriculus using the TRIzol reagent (Invitrogen, Carlsbad, CA). For 3'-RACE, first-strand cDNAs were synthesized from 5 µg total RNA using an adaptor primer supplied by the 3'-RACE system (Invitrogen). The reaction mixture was purified using a Wizard PCR preps DNA purification system (Promega Corp., Madison, WI) to remove excess bases, enzymes, and salts and was eluted in 50 µl sterilized water. One tenth of this purified cDNA served as a template for the PCR, using four degenerate sense-primers, based on the N-terminal seven-amino acid sequence of mammalian ghrelin (GSSFLSP), as follows: GRL-s7, 5'-GGGTCGAG(C/T)TTCTT(A/G)TC(A/G/T/C)-CC-3'; GRL-s8, 5'-GGGTCGAG(C/T)TTCTT(A/G)AG(C/T)CC-3'; GRL-s9, 5'-GGGTC-GAG(C/T)TTCCT(A/G/T/C)TC(A/G/T/C)CC-3'; and GRL-s10, 5'-GGGTCGAG(C/T)TT-CCT(A/G/T/C)AG(C/T)CC-3'. Primary PCR was performed using these degenerate sense-primers, a 3'-universal amplification primer, supplied with the 3'-RACE kit, and Ex Taq DNA polymerase (TaKaRa, Kyoto, Japan) as follows: 94 C for 1 min; 35 cycles at 94 C for 30 sec, 58 C for 30 sec, and 72 C for 1 min; and a final extension for 3 min at 72 C. After primary PCR, nested PCR was performed using one tenth of the primary PCR product purified by a Wizard PCR preps DNA purification kit (Promega Corp.) as starting material. Two degenerate nested sense-primers were designed, based on the amino acid sequence of the purified chicken ghrelin (SPTYKAI), as follows: 5'-TC(A/G/T/C)CC(A/G/T/C)AC(A/G/T/C)-TA(C/T)AA(A/G)AA(C/T)AT-3' for the chicken GRL-nest 1 primer and 5'-AG(C/T)CC-(A/G/T/C)AC(A/G/T/C)TA(C/T)AA(A/G)AA(C/T)AT-3' for the chicken GRL-nest 2 primer. The nested PCR was performed as follows: 94 C for 1 min; 30 cycles at 94 C for 30 sec, 46 C for 30 sec, and 72 C for 1 min; and a final extension for 3 min at 72 C. The putative ghrelin cDNA fragment was subcloned using a TOPO TA cloning kit (pCR II-TOPO vector, Invitrogen), and the nucleotide sequence was determined by automated sequencing (DNA sequencer, model 373; PE Applied Biosystems), according to the Thermosequence II dye terminator cycle sequencing kit protocol (Amersham Pharmacia Biotech) using the M13 forward and reverse primers.
5'-RACE
Poly (A)+ RNA from the chicken proventriculus was isolated using a mRNA purification kit (TaKaRa). First-strand cDNAs were synthesized from 500 ng poly (A)+ RNA with oligo-dT12–18 primer and SuperScript II reverse transcriptase (Invitrogen). The reaction mixture was purified using a Wizard PCR preps DNA purification system. One fifth of the purified cDNA was subjected to a TdT-tailing reaction of the 5'-ends of the first-strand cDNA with deoxycycidine triphosphate, according to the manufacturer’s protocol (Invitrogen). The resultant dC-tailed cDNA served as a template for primary 5'-RACE PCR. A gene-specific antisense primer, GRL-5'-1 (5'-GAAATAAAATAAGCCTACACG-3'), was designed, based on the partial sequence of the chicken ghrelin cDNA as determined by 3'-RACE. Primary PCR was performed using this gene specific primer, an abridged anchor primer supplied with the 5'-RACE kit, and Ex Taq DNA polymerase, as follows: 94 C for 1 min; 35 cycles at 94 C for 30 sec, 53 C for 30 sec, and 72 C for 1 min; and a final extension for 3 min at 72 C. The putative ghrelin fragment was subcloned using a TOPO TA cloning kit, and nucleotide sequences were determined as described above.
Northern blot analysis
Poly (A)+ RNA (2 µg) was prepared from 18 different tissues of broiler chicks and size-separated by electrophoresis on a denaturing 1% (wt/vol) agarose-formamide gel for 80 min under 50 V. RNA was then transferred onto a nylon membrane (Zeta-Probe; Bio-Rad Laboratories, Inc., Hercules, CA). The membrane was prehybridized in hybridization buffer [5x SSPE (750 mM NaCl, 50 mM NaH2PO4, and 5 mM EDTA, pH 7.4), 5x Denhardt’s solution, 50% formamide, 0.5% SDS, and 100 ng/ml calf thymus DNA] for 2 h at 37 C. Hybridization at 37 C for 24 h was performed with an EcoRI-digested chicken ghrelin cDNA 3'-RACE fragment (P8-n1–3, 
670 bp) labeled with [
-32P]deoxycycidine triphosphate using a megalabel DNA labeling kit (Amersham Pharmacia Biotech). After two washes with 2x SSC/0.1% SDS, one wash with 1x SSC/0.1% SDS, and one wash with 0.1x SSC/0.1% SDS at 55 C for 20 min each, the intensity of the hybridization signal was analyzed using a BAS-5000 bioimaging analyzer (Fuji Photo Film Co., Ltd., Tokyo, Japan).
Gene expression analysis by RT-PCR
Total RNA was extracted from 18 different tissues of broiler chicks. First-strand cDNAs were synthesized from 3 µg deoxyribonuclease I-treated (Invitrogen) total RNA, using SuperScript II reverse transcriptase (Invitrogen). PCR was performed using ReadyMix RED Taq PCR REACTION MIX with MgCl2 (Sigma, St. Louis, MO). The reaction mixtures (20 µl) contained 2 µl cDNA solution (300 ng total RNA equivalent), 0.5 µl 10-µM sense primer (5'-ATACAAGAAAACCAACAGCAAGAT-3', corresponding to nucleotides 266–289 of the chicken ghrelin cDNA), and 0.5 µl 10-µM antisense primer (5'-ACTAAGGAAGG-AAATAAAATAAGC-3', corresponding to nucleotides 687–710 of the chicken ghrelin cDNA). The reactions were performed as follows: 94 C for 10 min; 25 cycles at 94 C for 1 min, 51 C for 1 min, and 72 C for 1 min; and a final extension for 3 min at 72 C. The PCR products (445-bp) were analyzed by ethidium bromide staining on a 2% (wt/vol) agarose gel.
GH-releasing activity in rat
Male Sprague Dawley rats (250–300 g) were anesthetized with pentobarbital sodium throughout the experiment and cannulated in the femoral artery and vein. After sampling untreated blood from the femoral artery, a bolus of 20 ng/g of either synthetic rat ghrelin (n = 5) or chicken ghrelin-26 (n = 5) was injected into the femoral vein. Blood (150 µl) was continuously collected from each treated animal in a syringe containing EDTA (1 mg/ml blood), at 5, 10, 15, 20, 30, and 60 min after injection, and plasma was obtained by centrifugation at 5000 rpm for 5 min. Plasma GH levels were measured using a rat GH enzyme immunoassay system (Biotrak, Amersham Pharmacia Biotech). To evaluate an effect of dose or species of ghrelin, we carried out two-way ANOVA. A P value less than 0.05 was considered to be statistically significant.
Bioactivity in chicken
Eight-day-old chicks were divided into 7 groups. The control group received only vehicle (0.9% saline), whereas the 6 other groups were treated with either 0.4 µg, 2.0 µg, or 10.0 µg chicken ghrelin-26 or human ghrelin. Animals were killed by decapitation, and blood was collected from 10 animals at each time point: before (0 min) or 5, 10, 15, and 30 min after injection. The experimental protocol was approved by the ethical committee for animal experiments of the K. U. Leuven. Plasma GH levels were measured by RIA (34, 35). Corticosterone levels were analyzed using a commercial kit (ICN Pharmaceuticals, Inc., Asse-Relegem, Belgium), validated for use in the chicken (36). The sensitivity of both assays was 2 ng/ml, and the intra- and interassay coefficients of variation were 4.0% and 15.5%, respectively, for GH and 7.3% and 6.9%, respectively, for corticosterone. For evaluating the effect of ghrelin injection, we carried out one-way ANOVA at each time point, followed by Student’s t test or Cochran-Cox test for comparison of the mean. To evaluate effects of the time, dose, and species of ghrelin and their interactions, two-way ANOVA was applied. A P value less than 0.05 was considered to be statistically significant.
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). Ghrelin activity was observed in 38 sequential fractions (numbers 31–68). Those fractions were divided into 7 groups (A–G), according to the 7 peaks of activity. Each of these groups was subjected separately to purification by immunoaffinity chromatography. Peptides absorbed on the immunoaffinity column were initially separated by RP-HPLC on a µBondasphare C18 column. Active fractions were purified further by a second RP-HPLC step on a diphenyl or µBondasphare C18 column with a gentle gradient profile. Major peaks of active peptide were isolated from groups D (CM-HPLC fraction number 49–51, peak I; Fig. 2A) and G (CM-HPLC fraction number 65–68, peak II; Fig. 2B). Based on peak heights, the peptide yields were estimated to be 250 pmol for peak I and 270 pmol for peak II. Peptide sequence analysis showed that the peptides in peaks I and II were identical, containing 26 amino acid residues with the sequence: GSXFLSPTYKNIQQQKDTRKPTARLH (X, unidentified) (Fig. 2C). Because the N-terminal amino acid sequence of the purified peptides (GSXFLSP) is almost identical to the mammalian ghrelin consensus sequence (GSSFLSP), the chicken peptides were thus designated to be chicken ghrelin homologs. Based on mammalian and amphibian data, it was predicted that the unidentified amino acid residue at position 3 contains an acyl modification (1, 27).
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). An AATAAA polyadenylation signal is located in the 3'-UTR (nucleotides 814–819). A methionine, predicted to be the translation initiation site, is encoded by nucleotides 148–150. The deduced amino acid sequence of the coding region indicated that the chicken ghrelin precursor protein is composed of 116 amino acid residues (Fig. 4), of which the first 24 N-terminal residues were predicted to be a signal peptide, followed by the 26 amino acid residues of the ghrelin peptide. Two arginine residues, a typical dibasic processing signal, are present at the COOH-terminal end of the ghrelin peptide (nucleotides 295–300) (Fig. 3). The peptide sequence predicted from the cDNA was in agreement with the determined sequence of the purified peptide (Fig. 2C
). Based on the cDNA sequence, the unknown third amino acid residue was identified as a serine (Fig. 3). Thus, the complete amino acid sequence of chicken ghrelin was determined to be GSSFLSPTYKNIQQQKDTRKPTARLH.
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Tissue expression of chicken ghrelin
Northern blot analysis revealed a single ghrelin mRNA band (approximately 0.8 kb) that could only be detected in the proventriculus (Fig. 5A). To examine expression of the chicken ghrelin gene in other tissues, we performed RT-PCR analysis on total RNA extracted from 18 different tissues. In addition to high levels of expression in the proventriculus observed in Northern blot, moderate levels of gene expression were detected in the corpus striatum, and low levels were found in the cerebellum, optic lobes, brain stem, lung, spleen, duodenum, ileum, cecum, and rectum (Fig. 5B).
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). A similar stimulatory response was also observed in plasma GH levels of rats after iv injection of either chicken ghrelin-26 or rat ghrelin (Fig. 6B). Changes in plasma GH levels, by time, was apparent (P = 0.0005), and the levels increased 5 min after injection of either chicken or rat ghrelin, after which the increased levels returned to initial levels 60 min post injection. A difference of the effect of ghrelin species was not detected (P = 0.73)
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). Although neither an effect by time (P = 0.11) or dose (P = 0.79) was evident using two-way ANOVA, a significant increase in plasma GH levels was observed, 15 min after injection, at doses of 2.0 and 10.0 µg, compared with controls (which received saline). There was no effect of the type (chicken or human) of ghrelin used (P = 0.29 for 0.4-µg dose; P = 0.13 for 2.0-µg dose; P = 0.95 for 10.0-µg dose).
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). As shown in Fig. 8A, chicken ghrelin had both a time (P = 0.001) and dose (P = 0.02) effect. Plasma corticosterone levels significantly increased 5 min after injection of 2.0 and 10.0 µg ghrelin, compared with control values. Maximum increases in corticosterone levels were observed after 10–15 min, depending on the dose injected. The increased levels returned to initial levels 30 min after injection, except in the case of the 2.0- and 10.0-µg doses (where corticosterone levels remained somewhat higher). Human ghrelin also had a time (P = 0.02) and dose (P = 0.01) effect on corticosterone levels in chicken (Fig. 8B), and the changes were similar to those observed after chicken ghrelin administration. There was no specific ghrelin type effect on plasma corticosterone levels (P = 0.22 for 0.4-µg dose; P = 0.96 for 2.0-µg dose; P = 0.23 for 10.0-µg dose).
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Acylation and molecular form
A unique feature of ghrelin is the acylation, generally by n-octanoic acid, of a hydroxyl group on the third amino acid residue that is essential for ghrelin activity (1). In mammalian ghrelins, this modified amino acid is serine (1); whereas in amphibians, it is threonine (27). Like the mammalian species, the avian ghrelin has an n-octanoylated serine in the third position. Further studies are required to identify ghrelin, and its possible modification, in even lower vertebrates. The des-octanoyl form of ghrelin, des-acyl ghrelin, has no effect on intracellular calcium increase in GHSR62 cells (1) and does not bind to the GHS-R (37). Acylation of the third amino acid, whether it be serine or threonine, is a common feature of ghrelin in a wide variety of animals and is essential for its receptor binding, intracellular signaling, and biological activity.
In addition to the octanoylated form of ghrelin, we also identified a decanoylated form that comprised approximately 50% of the isolated chicken ghrelin. This finding is similar to those in human and bullfrog, in which the decanoylated form was found to represent 23% (Hosoda, H., et al., unpublished data) and 33% (27) of the total ghrelin population, respectively. In the rat, however, no decanoyl-modified ghrelin could be identified (Hosoda, H., et al., unpublished data). Recently, it has been reported that decanoylated ghrelin shows a potency similar to that of the octanoylated form in GHSR62 cells (38). The mechanisms involved in posttranslational modification of ghrelin are still unknown.
The mature, 26-residue chicken ghrelin is processed at a typical dibasic processing sequence, Arg-Arg, located at the C-terminal end of the ghrelin peptide. This sequence is similar also in the bullfrog (27) but not in mammals (1). It is interesting that the processing mechanism of ghrelin changed during evolution from avian to mammalian.
Relationship to the sequence and biological activity
The amino acid sequence of the chicken ghrelin precursor showed 38% and 27% sequence identity to its human and frog homologs, respectively. However, the sequence of the mature chicken ghrelin differs substantially from that of frog ghrelin (19% identity), while showing high homology to mammalian ghrelins (54% identity to human ghrelin), especially in the N-terminal seven residues (GSSFLSP). Indeed, amino acids at positions 1, 4, 5, 6, and 7 are conserved in all the species examined so far (27). In contrast, the sequence after the seventh amino acid varies substantially among species (27). The biological activity of chicken ghrelin is similar to that of rat ghrelin, as evaluated by its effects on intracellular calcium in GHSR62 cells and on plasma GH levels in vivo in the rat. In addition, chicken ghrelin increases plasma GH levels in chicks in vivo, with a potency similar to that of human ghrelin. These results are in agreement with the finding that the N-terminal tetrapeptide (GSSF), which retains the octanoyl group at Ser3, is the minimal active core of ghrelin (38, 39). Because the N-terminal tetrapeptide is identical among the chicken, human, and rat, similar biological activities are exhibited, even when heterologous systems were used. On the other hand, bullfrog ghrelin only weakly increases intracellular calcium concentrations in GHSR62 cells and is a poor inducer of GH release in rats, compared with the effects of rat ghrelin (27), presumably because of differences in the N-terminal portion of the peptide, including the acylated Thr3. In the bullfrog ghrelin, two of the four amino acids of the minimal core peptide differ from the human, rat, and chicken sequences, which probably reflects on ligand recognition by rat GHS-R (40). On the other hand, the role of the C-terminal sequence is still unknown. Human ghrelin (16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28) does not show any biological activity in GHSR62 cells (38). Bowers (41) has suggested that both acylation and the C-terminal part of the ghrelin molecule play a role in establishing the bioactive conformation of the intact acylated ghrelin molecule.
In chicks, plasma corticosterone levels increase significantly after chicken or human ghrelin administration. The increase has been observed with a much smaller dose than that used in human studies (26). Although the mechanisms by which ghrelin increases plasma corticosterone levels are not known yet, it is possible that hypophyseal ACTH is involved (26). This result suggests that, in addition to its GH-releasing activity, ghrelin may be an important regulator of adrenal function in birds.
Expression sites of chicken ghrelin mRNA
In the chicken, ghrelin is predominantly synthesized in the proventriculus, which is comparable with the gastric fundus. On the other hand, no ghrelin mRNA expression could be detected in the gizzard, which is a part of the avian stomach that is only involved in the mechanical processing of the ingested food. These results are consistent with the fact that the gastric fundus is the major ghrelin-producing site in the rat and human (1, 18, 42, 43). In both the chicken and rat, ghrelin (produced and secreted from the stomach) is likely to act directly on the pituitary via the systemic circulation. In addition, indirect pathways have also been proposed, such as one through a hypothalamic U-factor (16, 29, 30, 41). In addition to the stomach, ghrelin is also synthesized in the rat brain. A few ghrelin-producing cells are located in the hypothalamic arcuate nucleus (1) and probably participate in the regulation of GH secretion from the pituitary (3) as well as feeding behavior (5, 14, 15, 16, 17). In the present study, moderate or low levels of ghrelin mRNA were observed in the corpus striatum, cerebellum, optic lobe, and brain stem of the chick, suggesting direct action of hypothalamic ghrelin on pituitary function. Furthermore, ghrelin affects feeding behavior in chicks; an intracerebroventricular bolus injection of rat ghrelin inhibits food intake (32). To identify other possible functions of ghrelin mediated by the central nervous system, it will be important to identify the detailed distribution of ghrelin-producing cells in the chicken brain. In addition to the proventriculus and the brain, chicken ghrelin mRNA is also expressed in the lung, spleen, duodenum, ileum, cecum, and rectum, although at very low levels. This distribution pattern corresponds to the pattern of GHS-R expression (data not shown), suggesting an autocrine and/or paracrine action of ghrelin in those organs.
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Acknowledgments |
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Footnotes |
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Abbreviations: ACN, Acetonitrile; AcOH, acetic acid; [Ca2+]i, intracellular calcium concentration; CHO, Chinese hamster ovary; CM, carboxymethyl; FLIPR, fluorometric imaging plate reader; GHS, GH secretagogue; GHS-R or GHSR, GHS receptor; RACE, rapid amplification of the cDNA ends; RP, reverse-phase; TFA, trifluoroacetic acid.
Received March 4, 2002.
Accepted for publication May 7, 2002.
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