Molecular and Functional Characteristics of APJ TISSUE DISTRIBUTION OF mRNA AND INTERACTION WITH THE ENDOGENOUS LIGAND APELIN*

We have recently identified apelin as the endogenous ligand for human APJ. In rats, the highest expression of APJ mRNA was detected in the lung, suggesting that APJ and its ligand play an important role in the pulmonary system. When apelin-36 and its pyroglutamylated C-terminal peptide, [ < Glu 65 ]apelin-13, were compared in microphysiometric analyses, the elevation of extracellular acidification induced in cells expressing APJ by [ < Glu 65 ]apelin-13 was transient, whereas that by ape-lin-36 was sustained. These responses were almost completely inhibited by a specific inhibitor for G i or that for Na 1 /H 1 exchanger. 125 I -Labeled [ < Glu 65 ]apelin-13 analogue specifically bound to APJ with a high affinity, and [ < Glu 65 ]apelin-13 was more potent than apelin-36 in competitive inhibition assays. Because pretreatment with apelin-36 but not [ < Glu 65 ]apelin-13 drastically re-duced the binding of the labeled apelin to APJ, the different patterns of acidification induced by these two peptides appeared to reflect their dissociation rather than association with APJ. Apelin elicited the migration of APJ-expressing cells, and [ < Glu 65 ]apelin-13 was more potent

ever, the molecular and functional characteristics of APJ have not been analyzed enough, because its endogenous ligand is still unidentified.
Although the first demonstration of identifying orphan 7TMR ligands was the discovery of orphanin/nociceptin (5, 6), we have recently established a strategy widely applicable for identifying orphan 7TMR ligands by detecting signal transductions in CHO cells expressing orphan 7TMRs (2,7,8). By the application of this strategy, we found that the peptide-enriched fractions prepared from bovine stomach tissues showed activities to promote specifically extracellular acidification in CHO cells expressing APJ in the microphysiometric assay, and we then succeeded in identifying an endogenous peptidic APJ ligand. In addition, we isolated bovine, human, rat, and mouse cDNAs encoding the ligand peptide (2,9). We named the ligand peptide "apelin" after APJ endogenous ligand (2).
In this study, we isolated a rat APJ cDNA and analyzed the distribution of its mRNA in rat tissues. We subsequently analyzed functional differences in the interaction with APJ between apelin-36 and [ϽGlu 65 ]apelin-13 and found that the N-terminal portion of apelin-36 could modulate the interaction with APJ, although an essential structure for binding localized in the C-terminal portion. In addition, We demonstrated that apelin shows a chemotactic action and that heterogeneous molecular forms of apelin including shorter forms corresponding to [ϽGlu 65 ]apelin-13 are produced in vivo in bovine colostrum.

EXPERIMENTAL PROCEDURES
Cloning of Rat APJ cDNA-Rat APJ cDNA fragments were isolated from poly(A)ϩ RNA of rat brain by the 5Ј and 3Ј rapid amplification of cDNA ends (RACE) method using a Marathon cDNA amplification kit (CLONTECH, Palo Alto, CA). Primers used were 5Ј-GACAAAGAT-GAGGTAGCTGCTGAG-3Ј (F1) and 5Ј-GTCGAGCGTTAGCCACTG-GCC-3Ј (F2) for 5Ј-RACE and 5Ј-TGTTACTTCTTCATTGCCCAAAC-CAT-3Ј (R1), 5Ј-TGGGGTGTCCTCCACTGCTGT-3Ј (R2), and 5Ј-ACT-CAGAGTGGGCCTGGGAGG-3Ј (R3) for 3Ј-RACE. First PCR was carried out using F2 for 5Ј-RACE or R3 for 3Ј-RACE in combination with the adapter primer 1 provided with the kit in a 25-l reaction mixture prepared with appropriately diluted cDNAs, 0.2 M primers, 1.25 units of ExTaq DNA polymerase (Takara Shuzo, Kyoto, Japan) treated with TaqStart antibody (CLONTECH), 0.1 mM dNTPs, and the reaction buffer supplemented with the polymerase. Amplification in the first PCR was conducted under the following conditions: at 94°C for 2 min for the denaturation of the template and the activation of ExTaq DNA polymerase; 5 cycles at 98°C for 10 s and at 72°C for 2 min; 5 cycles at 98°C for 10 s and at 70°C for 2 min; and 25 cycles at 98°C for 10 s and at 68°C for 2 min. The second PCR was carried out using F1 and F2 for 5Ј-RACE or R1, R2 and R3 for 3Ј-RACE with 1.0 l of the * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBank TM /EBI Data Bank with accession number AB033170.
‡ To whom correspondence should be addressed. Tel.: 81-298-64-5035; Fax: 81-298-64-5000; E-mail: Hinuma_Shuji@takeda.co.jp. 1 The abbreviations used are: 7TMR, seven-transmembrane-domain receptor; PCR, polymerase chain reaction; RT, reverse transcription; CHO, Chinese hamster ovary; PTX, pertussis toxin; MIA, methyl-isobutyl amirolide; RACE, rapid amplification of cDNA ends; HPLC, high performance liquid chromatography; BSA, bovine serum albumin. reaction mixture from the first PCR in combination with adapter primers 1 and 2 provided with the kit, and the final amplification step was elongated to 33 cycles. DNA sequences of cDNA fragments amplified were determined with a model 377 DNA sequencer (PE Biosystems) and analyzed with the computer software DNASIS (Hitachi Software Engineering, Yokohama, Japan). A cDNA fragment with the entire open reading frame for expression vector construction was amplified from rat heart cDNA with a primer set (5Ј-AAGCACCCTCAGACCACT-TACTC-3Ј and 5Ј-TTTGCAAGGCTCCTTCCCTTTCC-3Ј) based on the sequences obtained from the above cDNA fragments. PCR was carried out in a 25-l reaction mixture prepared with appropriately diluted cDNAs, 0.2 M primers, 1.25 units of KlenTaq DNA polymerase (CLON-TECH) treated with TaqStart antibody (CLONTECH), 0.1 mM dNTP, and the reaction buffer supplemented with the polymerase. Amplification was conducted under the following conditions: at 94°C for 2 min for the denaturation of the template and the activation of KlenTaq DNA polymerase; 30 cycles at 98°C for 10 s and at 68°C for 30 s for amplification; and at 72°C for 1 min for extension.
Preparation of CHO Cells Expressing APJ-CHO cells expressing human APJ (CHO-A10) were established as described previously (2). CHO cells expressing rat APJ were prepared principally by the same method.
Quantitative Analyses for Rat APJ mRNA by Reverse Transcription (RT)-PCR-Poly(A)ϩ RNAs were prepared from the tissues of adult (8 -12-week-old) and neonate (0 -3-day-old) Wistar rats as described previously (9). We quantified rat APJ mRNA by means of a Prism 7700 sequence detector (PE Biosystems) with a primer set (5Ј-CCACCTGGT-GAAGACTCTCTACA-3Ј and 5Ј-TGACGTAACTGATGCAGGTGC-3Ј) and a probe labeled with fluorescent dyes (5Ј-(FAM)-TGACAGCTTC-CTCATGAATGTCTTTCCC-(TAMRA)-3Ј). RT-PCR was carried out in a 25-l reaction mixture prepared with a TaqMan PCR core reagent kit (PE Biosystems) containing an appropriately diluted cDNA solution, 0.2 M each primer, and 0.1 M probe. PCR was performed under the following conditions: at 50°C for 10 min for the reaction of uracil-Nglycosylase to prevent the amplification of PCR products carried over; at 95°C for 2 min for the activation of AmpliTaq Gold DNA polymerase; and 43 cycles at 95°C for 15 s and at 55°C for 90 s for the amplification. The quantification of APJ mRNA in neonatal tissues was carried out principally according to the conditions described above, but amplification was performed by 45 cycles of 95°C for 15 s and at 57°C for 90 s. In order to obtain a calibration curve, we amplified the known amount of a cDNA fragment of rat APJ in the same manner as the samples. A good linear relationship was obtained between the amount of rat APJ cDNA input and the release of the reporter dye within the range of 10 to 10 6 copies. Rat glyceraldehyde-3-phosphate dehydrogenase mRNA was also measured as an internal control as described previously (9).
Microphysiometric Assays-Extracellular acidification rates were measured with a Cytosensor (Molecular Devices Corp.) as described previously (2). CHO cells expressing human or rat APJ were dispersed with trypsin, and the cells were dispensed into cell capsules (Molecular Devices) at 2.7 ϫ 10 5 cells/capsule and cultured overnight. Then, each cell capsule was set to the device, and the cells were continuously loaded with a low-buffered RPMI 1640 medium (Molecular Devices) until the rate of acidification became stable. The acidification rates were measured every 120 s (flow on at 100 l/min for 80 s; flow off for 8 s; measuring acidification rates for 30 s). For the treatment of pertussis toxin (PTX), CHO-A10 cells were seeded at 9 ϫ 10 4 cells/capsule and cultured overnight, and then 100 ng/ml PTX (P-9452, Sigma) was added to the culture 24 h before setting the capsules to the device. Methylisobutyl amirolide (MIA) (Research Biochemicals Inc., MA, USA) was dissolved in the low-buffered RPMI 1640 medium at 10 M and exposed to the cells just before adding samples.
[ϽGlu 65 ,Nle 75 ,Tyr 77 ]apelin-13 was radioiodinated with Na 125 I (IMS-30, Amersham Pharmacia Biotech) by a method using lactoperoxidase (Sigma) as described elsewhere (10). After the reaction, the labeled and unlabeled peptides were separated by reversed-phase HPLC. Aliquots of the labeled peptide were stored at Ϫ30°C until used.
Receptor Binding Assays Using Membrane Preparations-The membrane fractions of CHO-A10 cells prepared by the method as described previously (11) were incubated with [ 125 I][ϽGlu 65 ,Nle 75 ,Tyr 77 ]apelin-13 in 100 l of the binding buffer containing 0.1% bovine serum albumin (BSA) in 96-well microplates (Serocluster, Corning Costar Corp.) at room temperature for 90 min. In order to determine the amounts of nonspecific binding, unlabeled [ϽGlu 65 ,Nle 75 ,Tyr 77 ]apelin-13 was simultaneously added to the wells. After incubation, bound and free radioactivities were separated through rapid filtration using the glassfiber filter units (GF/C, Packard Instrument Co.) of a 96-well cell harvester (Packard). The filter units were completely dried, and Microcinti O (Packard) was added to each well. The radioactivity of each well was counted with a TopCount liquid scintillation counter (Packard). The dissociation constant (K d ) and the number of binding sites (B max ) were determined by the method of Scatchard (12).
Receptor Binding Assays Using Intact Cells-CHO-A10 cells were seeded at 1 ϫ 10 5 cells/well in 24-well tissue culture plates and grown for 2 days. Prior to the binding experiments, the cells were washed three times with Hanks' balanced salt solution containing 0.05% BSA. In order to determine the amount of nonspecific binding, 1 M unlabeled [ϽGlu 65 ,Nle 75 ,Tyr 77 ]apelin-13 was added to the wells. The cells were incubated with 200 pM [ 125 I][ϽGlu 65 ,Nle 75 ,Tyr 77 ]apelin-13 for the time desired at room temperature. After the incubation, the cells were washed four times with Hanks' balanced salt solution containing 0.05% BSA and then lysed with 0.2 N NaOH containing 1% SDS. The radioactivity of the cell lysate was measured with a gamma counter (Beckman). The binding of radiolabeled apelin to the cells after exposure to unlabeled apelin was determined as follows. CHO-A10 cells were incubated with 1 mM [ϽGlu 65 ]apelin-13 or apelin-36 for 90 min and then washed four times with Hanks' balanced salt solution containing 0.05% BSA to remove unbound peptides. After the cells were incubated with the radiolabeled apelin, the amount of the labeled apelin bound to the cells was determined as described above.
Chemotactic Assays-Chemotactic assays were performed with 96well microchemotaxis chambers (Neuro Probe). [ϽGlu 65 ]apelin-13 and apelin-36 were diluted with Dulbecco's modified minimum essential medium supplemented with 0.5% BSA (Dulbecco's modified minimum essential medium/BSA), and 37 l of each diluted solution was added to separate lower chambers. A polyvinylpyrrolidone-free polycarbonateframed filter with 5-m pores (Neuro Probe), after being precoated with 10 g/ml bovine fibronectin (Yagai Research Center, Yamagata, Japan), was used to separate the upper and lower chambers. CHO-A10 cells and mock-transfected CHO cells were harvested and suspended in Dulbecco's modified minimum essential medium/BSA. Cell suspensions at 1 ϫ 10 5 cells/200 l/well were added to the upper chamber. The chemotaxis chamber was incubated at 37°C for 4 h in a CO 2 incubator with 5% CO 2 in air. After the nonmigrating cells on the upper surface of the filter were scraped off, those migrating to the bottom of the filter were fixed and stained with Diff-Quick (International Reagent Corporation, Hyogo, Japan), and the absorbance at 570 nm was measured with a Benchmark microplate reader (Bio-Rad).
Gel Filtration Analysis of Bovine Colostrum-Colostrum from Holstein cows was boiled for 15 min, supplemented with up to 1 M acetic acid, and homogenized using a Polytron homogenizer. The clear supernatant prepared by centrifugation was fractionated as described previously (2). In brief, the fraction eluted with 30% acetonitrile in C 18 open column (Prep C 18 , Waters) chromatography was applied to HiPrep CM-Sepharose FF column (Amersham Pharmacia Biotech). The eluate with 0.5 M ammonium acetate was treated with acetone, and desalted with Sep-Pak C 18 column (Waters). After lyophilization, this fraction was applied on Superdex Peptide gel filtration column (Amersham Pharmacia Biotech) chromatography. Synthetic apelin-36 and [ϽGlu 65 ]apelin-13 were applied on the same chromatography in order to determine fraction in which they were eluted. Apelin present in each fraction was detected on the basis of the cAMP production-inhibitory activity on CHO-A10 cells stimulated with forskolin as described previously (9).

RESULTS
Cloning of Rat APJ cDNA-A rat APJ cDNA was isolated from rat brain poly(A) ϩ RNA. The cDNA encoded a protein with a length of 377 amino acids. The deduced amino acid sequences of rat and human APJ are aligned in Fig. 1. Amino acid identity between the two sequences was 87.2%. We confirmed that CHO cells expressing rat APJ as well as those expressing human APJ specifically responded to [ϽGlu 65 ]apelin-13 in a dose-dependent manner in the microphysiometric assay (data not shown).
Tissue Distribution of Rat APJ mRNA-We analyzed the precise distribution of APJ mRNA in rat tissues by a quantitative RT-PCR method. As shown in Fig. 2, we detected APJ mRNA in almost all tissues tested, although their quantity varied considerably among the tissues. The highest expression was detected in the lungs of infants, and a comparable level of expression was also detected in those of adults. Interestingly, APJ mRNA expression tended to be higher in infant tissues (e.g. the kidney, stomach, and intestine) than those in adults. In the peripheral tissues of adults, moderate levels of expression were widely detected in the heart, thyroid gland, kidney, adrenal gland, adipose, ovary, uterus, femur, costal cartilage, and placenta. Similar levels of expression were also detected in the central nervous system in adults, such as the hypothalamus, medulla oblongata, and spinal cord. In these experiments, the levels of glyceraldehyde-3-phosphate dehydrogenase mRNA expression were almost consistent among the tissues, within the range of 0.7 ϫ 10 5 to 9.1 ϫ 10 5 copies/ng of poly(A) ϩ RNA except for pituitary, heart, and mammary gland (1.1 ϫ 10 6 to 2.2 ϫ 10 6 copies/ng of poly(A) ϩ RNA) and skeletal muscle (4.6 ϫ 10 6 copies/ng of poly(A) ϩ RNA).
Analyses for Extracellular Acidification Induced by Apelin-We exposed CHO-A10 cells with [ϽGlu 65 ]apelin-13 and human apelin-36 for a relatively long time (7 min 2 s). As shown in Fig. 3 Poly(A) ϩ RNA preparations obtained from the indicated rat tissues were subjected to quantitative RT-PCR analyses using a Prism 7700 sequence detector. Poly(A) ϩ RNAs of the placenta and mammary gland were prepared from female rats 17 and 21 days into pregnancy, and those of neonatal tissues were from rats of 0 to 3 days after birth, respectively. changes in the acidification rate after removal of the samples were quite distinctive between [ϽGlu 65 ]apelin-13 and apelin-36; the elevated acidification rates induced by apelin-36 at 10 to 100 nM were kept at a high level even after 20 cycles.
In order to compare intracellular mechanisms by which [ϽGlu 65 ]apelin-13 and apelin-36 caused extracellular acidification in CHO-A10 cells, we examined the effects of enzyme inhibitors. Because apelin could effectively inhibit the forskolin-stimulated cAMP production in CHO-A10 cells (2,9), APJ was thought to couple to the inhibitory G protein, G i . We thus first tested the effects of PTX treatment. As shown in Fig. 4, both [ϽGlu 65 ]apelin-13 and apelin-36 actions were obviously suppressed by the PTX treatment, suggesting that the signal transduction pathways stimulated by both peptides were transduced via G i . On the other hand, when we treated the CHO-A10 cells with MIA, the specific inhibitor for Na ϩ /H ϩ exchanger (13), the acidification rates promoted by both peptides were also evidently suppressed (Fig. 5), suggesting that they induced the promotion of the extracellular acidification through the Na ϩ /H ϩ exchanger. These results using the two inhibitors indicate that the promotion of acidification induced by both peptides is caused through essentially the same signal transduction pathways, although the profiles of acidification induced by the two peptides were considerably different. Chemotactic Action of Apelin-As a functional characterization of APJ, we examined the chemotactic action of apelin. As shown in Fig. 9, [ϽGlu 65 ]apelin-13 showed a potent chemotaxis-inducing activity to CHO-A10 cells. Apelin-36 also induced the chemotactic movement of the cells; however, its potency was weaker than [ϽGlu 65 ]apelin-13. The dose-response curves for [ϽGlu 65 ]aplein-13 and apelin-36 were typically bell-shaped. The two peptides did not show the chemotactic activity to mock-transfected CHO cells. With checkerboard analysis, the addition of apelin in the upper chamber did not induce migration and inhibited the migration toward the ligand in the lower chamber (data not shown), indicating that the migration in response to apelin was chemotactic but not chemokinetic.
Gel Filtration Analysis for Molecular Forms of Apelin in Bovine Colostrum-A peptide-enriched fraction was prepared from bovine colostrum by a combination of C 18 reversed phase and CM-Sepharose ion exchange column chromatographies. In order to analyze the molecular forms of endogenous apelin, we subjected this fraction to gel filtration, and biologically active apelin contained in each fraction was detected by the cAMP production-inhibitory assay utilizing CHO-A10 cells. As shown in Fig. 10, two peaks of the activities were detected at positions corresponding to those of synthetic apelin-36 and [ϽGlu 65 ]apelin-13 eluted. However, these fractions did not show such activities on mock-transfected CHO calls (data not shown). These results indicate that both the long and short forms of apelin, corresponding to apelin-36 and [ϽGlu 65 ]apelin- 13, respectively, are produced at least in bovine colostrum, although the long forms of apelin appeared to be dominant. DISCUSSION By isolating a rat APJ cDNA, we found that the primary structure of APJ is highly conserved between human and rat. The amino acid sequences of mature apelin peptides (e.g. apelin-36) are also highly conserved among human, bovine, rat, and mouse (2,9), suggesting that the structures of APJ and apelin are highly conserved in evolution. In quantitative RT-PCR analyses, the highest level of APJ mRNA expression was detected in the lung in rat tissues. In our recent study, a very high level of apelin mRNA has been also detected in the lung (9). Taken together, it is suggested that APJ and its ligand play a crucial role in the pulmonary system in rats. However, in human tissues, APJ mRNA is very highly expressed in the spleen, but its expression level is low in the lung (14). These results might reflect the functional differences of APJ between the two species. The expression of APJ mRNA in infant rats was higher than that in adults. Because the expression level of rat apelin mRNA was also high in infants, 2 APJ and apelin were expected to have a regulatory function in the process of development.
Bovine, human, rat, and mouse apelin cDNAs encode preproproteins with the same length of 77 amino acids (2,9). Based on the analyses for the endogenous apelin purified from the bovine stom-ach, a mature peptide (apelin-36) has been found to consist of 36 amino acid residues, starting from Leu 42 and running to the C terminus of Phe 77 . Because there are many basic amino acid residues in apelin-36 (i.e. lysine 72 and arginine 46, 49, 59, 60, 63, 64, 66, and 68) as potential proteolytic cleavage sites, we presumed that shorter forms of apelin might exist. Indeed, synthetic apelin-13, [ϽGlu 65 ]apelin-13, and apelin-17 showed stronger extracellular acidifying activities than apelin-36 to CHO-A10 cells in dose-response analyses. We synthesized [ϽGlu 65 ]apelin-13 as a pyroglutamylated form of apelin-13, and the two peptides were equipotent in the acidification rate promoting activity (2). Because [ϽGlu 65 ]apelin-13 is structurally more stable than apelin-13, we have mainly used [ϽGlu 65 ]apelin-13 as a representative short form of apelin. In this study, we attempted to reveal functional differences in heterogeneous apelin molecules, and we found that the induction of the extracellular acidification in CHO-A10 cells was quite different between [ϽGlu 65 ]apelin-13 and apelin-36. Somatostatin exists in multiple forms (i.e. somatostatin-14, somatostatin-25, and somatostatin-28) (15). We compared somatostatin-14 and somatostatin-28 in the microphysiometric assay with CHO cells somatostatin type 2 receptor, but we could not detect any obvious differences between the two forms in the induction of extracellular acidification (data not shown).
We have reported that apelin is abundantly produced in milk and colostrum and that its molecular form is fairly heterogeneous (9). We thus speculate that apelin would act on neonates through the oral intake of the colostrum and milk. By purification, we have confirmed that at least three different apelin isoforms, starting at the N-terminal amino acid residues Leu 42 , Gly 47 , and Ser 50 (which would correspond to apelin-36, -31, and -28), exist in bovine milk (9). In this study, we analyzed whether short forms of apelin corresponding to [ϽGlu 65 ]apelin-13 were actually produced in vivo. Because apelin is known to be the most abundant in colostrum, we analyzed the molecular heterogeneity of endogenous apelin in bovine colostrum by gel filtration and found that both long and short forms of apelin, corresponding to apelin-36 and [ϽGlu 65 ]apelin-13, respectively, were produced in bovine colostrum. In addition, we have observed that apelin-13 was actually produced when apelin cDNA was expressed in CHO cells (9). We therefore consider was thus supposed to rapidly dissociate from APJ so that the labeled ligand could replace with [ϽGlu 65 ]apelin-13 bound to APJ. In contrast, the labeled ligand scarcely bound to CHO-A10 cells pretreated with apelin-36, suggesting that apelin-36 bound hardly dissociates from APJ. In our preliminary experiments, the binding of the labeled apelin to CHO-A10 cells pretreated with apelin-36 was significantly recovered after treating the cells with acid (data not shown), suggesting that the decrease of the labeled apelin binding caused by the treatment with apelin-36 is due neither to desensitization nor to internalization in APJ. Although a precise comparison of association rate constants by utilizing the corresponding radioligands of [ϽGlu 65 ]apelin-13 and apelin-36 will be necessary, we believe that the prolonged acidification caused by aplein-36 in the microphysiometric assays reflects characteristics in its dissociation from APJ. Alternatively, our results indicate that the N-terminal portion of apelin-36 modulates the dissociation from the receptor.
In this study, we demonstrated that apelin peptides showed chemotactic activities to CHO-A10 cells. In the chemotactic assay, [ϽGlu 65 ]apelin-13 was more potent than apelin-36. As we have demonstrated previously, [ϽGlu 65 ]apelin-13 also shows higher activity than apelin-36 in the cAMP productioninhibitory assay using CHO-A10 cells (9). It should be noticed that in the both assays CHO-A10 cells are exposed consistently to test samples during assays. In such cases, the potency of an apelin peptide would be determined by its association rate rather than dissociation rate against APJ. Alternatively, in cases such as the in vivo situation, if APJ is transiently exposed to an apelin peptide, apelin-36 might show greater activity than [ϽGlu 65 ]apelin-13. In any case, further studies are nec-essary to confirm whether apelin physiologically acts as one of chemotactic factors.
In this study, we demonstrated that the N-terminal portion of apelin-36 is very important to modulate the interaction with APJ, although the core structure of apelin to bind the receptor is situated in the C-terminal portion. Because both the long and short forms of apelin are produced in vivo under some conditions, they might play different physiological roles, respectively, in vivo as well as in vitro. FIG. 10. Gel filtration chromatography of bovine colostrum. The peptideenriched fraction prepared from bovine colostrum was analyzed by gel filtration chromatography. Apelin contained in each fraction was detected by the forskolin-stimulated cAMP production-inhibitory assay. The positions of synthetic apelin-36 and [ϽGlu 65 ]apelin-13 eluted in the same chromatography are indicated with arrows in the figure.