Title: Molecular and Functional Characteristics of APJ
Abstract: 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, [<Glu65]apelin-13, were compared in microphysiometric analyses, the elevation of extracellular acidification induced in cells expressing APJ by [<Glu65]apelin-13 was transient, whereas that by apelin-36 was sustained. These responses were almost completely inhibited by a specific inhibitor for Gi or that for Na+/H+ exchanger. 125I -Labeled [<Glu65]apelin-13 analogue specifically bound to APJ with a high affinity, and [<Glu65]apelin-13 was more potent than apelin-36 in competitive inhibition assays. Because pretreatment with apelin-36 but not [<Glu65]apelin-13 drastically reduced 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 [<Glu65]apelin-13 was more potent than apelin-36 in this activity. Heterogeneous molecular forms of apelin corresponding to apelin-36 and [<Glu65]apelin-13 were produced in bovine colostrum. Apelin-36 and [<Glu65]apelin-13 might have different functions in vivo and in vitro. 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, [<Glu65]apelin-13, were compared in microphysiometric analyses, the elevation of extracellular acidification induced in cells expressing APJ by [<Glu65]apelin-13 was transient, whereas that by apelin-36 was sustained. These responses were almost completely inhibited by a specific inhibitor for Gi or that for Na+/H+ exchanger. 125I -Labeled [<Glu65]apelin-13 analogue specifically bound to APJ with a high affinity, and [<Glu65]apelin-13 was more potent than apelin-36 in competitive inhibition assays. Because pretreatment with apelin-36 but not [<Glu65]apelin-13 drastically reduced 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 [<Glu65]apelin-13 was more potent than apelin-36 in this activity. Heterogeneous molecular forms of apelin corresponding to apelin-36 and [<Glu65]apelin-13 were produced in bovine colostrum. Apelin-36 and [<Glu65]apelin-13 might have different functions in vivo and in vitro. seven-transmembrane-domain receptor polymerase chain reaction reverse transcription Chinese hamster ovary pertussis toxin methyl-isobutyl amirolide rapid amplification of cDNA ends high performance liquid chromatography bovine serum albumin APJ, a member of the seven-transmembrane-domain receptor (7TMR)1 family, was originally isolated from human genomic DNA by polymerase chain reaction (PCR) (1.O'Dowd B.F. Heiber M. Chan A. Heng H.H.Q. Tsui L.-C. Kennedy J.L. Shi X. Petronis A. George S.R. Nguyen T. Gene. 1993; 136: 355-360Crossref PubMed Scopus (655) Google Scholar). Despite the significant structural relationship between angiotensin II receptor and APJ (i.e. 30–40% identity in amino acid sequences), angiotensin II did not interact with APJ expressed in fibroblasts (1.O'Dowd B.F. Heiber M. Chan A. Heng H.H.Q. Tsui L.-C. Kennedy J.L. Shi X. Petronis A. George S.R. Nguyen T. Gene. 1993; 136: 355-360Crossref PubMed Scopus (655) Google Scholar) and Chinese hamster ovary (CHO) cells (2.Tatemoto K. Hosoya M. Habata Y. Fujii R. Kakegawa T. Zou M.-X. Kawamata Y. Fukusumi S. Hinuma S. Kitada C. Kurokawa T. Onda H. Fujino M. Biochem. Biophys. Res. Commun. 1998; 251: 471-476Crossref PubMed Scopus (1289) Google Scholar). APJ had been thus originally classified into a ligand-unknown “orphan” 7TMR. On the other hand, APJ reportedly acts as one of candidate coreceptors, together with CD4, in the process of human immunodeficiency virus type 1 infection (3.Choe H. Farzan M. Konkel M. Martin K. Sun Y. Marcon L. Cayabyab M. Berman M. Dorf M.E. Gerard N. Gerard G. Sodroski J. J. Virol. 1998; 72: 6113-6118Crossref PubMed Google Scholar, 4.Hoffman T.L. Stephens E.B. Narayan O. Doms R.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11360-11365Crossref PubMed Scopus (100) Google Scholar). However, 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.Reinscheid R.K. Nothacker H.P. Bourson A. Ardati A. Henningsen R.A. Bunzow J.R. Grandy D.K. Langen H. Monsma F.J. Civelli O Science. 1995; 270: 792-794Crossref PubMed Scopus (1752) Google Scholar, 6.Meunier J.-C. Mollereau C. Toll L. Saudeau C. Moisand C. Alvinerie P. Butour J.-L. Guillemot J.-C. Ferrara P. Monsarrat B. Mazarguil H. Vassart G. Parmentier M. Constetin J. Nature. 1995; 377: 532-535Crossref PubMed Scopus (1796) Google Scholar), we have recently established a strategy widely applicable for identifying orphan 7TMR ligands by detecting signal transductions in CHO cells expressing orphan 7TMRs (2.Tatemoto K. Hosoya M. Habata Y. Fujii R. Kakegawa T. Zou M.-X. Kawamata Y. Fukusumi S. Hinuma S. Kitada C. Kurokawa T. Onda H. Fujino M. Biochem. Biophys. Res. Commun. 1998; 251: 471-476Crossref PubMed Scopus (1289) Google Scholar, 7.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. Nature. 1998; 393: 272-276Crossref PubMed Scopus (526) Google Scholar, 8.Hinuma S. Onda H. Fujino M. J. Mol. Med. 1999; 77: 495-504Crossref PubMed Scopus (54) Google Scholar). 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.Tatemoto K. Hosoya M. Habata Y. Fujii R. Kakegawa T. Zou M.-X. Kawamata Y. Fukusumi S. Hinuma S. Kitada C. Kurokawa T. Onda H. Fujino M. Biochem. Biophys. Res. Commun. 1998; 251: 471-476Crossref PubMed Scopus (1289) Google Scholar, 9.Habata Y. Fujii R. Hosoya M. Fukusumi S. Kawamata Y. Hinuma S. Kitada C. Nishizawa N. Murosaki S. Kurokawa T. Onda H. Tatemoto K. Fujino M. Biochim. Biophys. Acta. 1999; 1452: 25-35Crossref PubMed Scopus (303) Google Scholar). We named the ligand peptide “apelin” after APJ endogenousligand (2.Tatemoto K. Hosoya M. Habata Y. Fujii R. Kakegawa T. Zou M.-X. Kawamata Y. Fukusumi S. Hinuma S. Kitada C. Kurokawa T. Onda H. Fujino M. Biochem. Biophys. Res. Commun. 1998; 251: 471-476Crossref PubMed Scopus (1289) Google Scholar). Although a mature apelin peptide consists of 36 amino acid residues (apelin-36), we found that the shorter C-terminal peptides, with a length of 13 amino acids (apelin-13) and its N-terminal pyroglutamylated form, [<Glu65]apelin-13, showed higher acidification rate-promoting and cAMP production-inhibitory activities than apelin-36 (2.Tatemoto K. Hosoya M. Habata Y. Fujii R. Kakegawa T. Zou M.-X. Kawamata Y. Fukusumi S. Hinuma S. Kitada C. Kurokawa T. Onda H. Fujino M. Biochem. Biophys. Res. Commun. 1998; 251: 471-476Crossref PubMed Scopus (1289) Google Scholar). 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 [<Glu65]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 [<Glu65]apelin-13 are produced in vivo in bovine colostrum. Rat APJ cDNA fragments were isolated from poly(A)+ RNA of rat brain by the 5′ and 3′ rapid amplification of cDNA ends (RACE) method using a Marathon cDNA amplification kit (CLONTECH, Palo Alto, CA). Primers used were 5′-GACAAAGATGAGGTAGCTGCTGAG-3′ (F1) and 5′-GTCGAGCGTTAGCCACTGGCC-3′ (F2) for 5′-RACE and 5′-TGTTACTTCTTCATTGCCCAAACCAT-3′ (R1), 5′-TGGGGTGTCCTCCACTGCTGT-3′ (R2), and 5′-ACTCAGAGTGGGCCTGGGAGG-3′ (R3) for 3′-RACE. First PCR was carried out using F2 for 5′-RACE or R3 for 3′-RACE in combination with the adapter primer 1 provided with the kit in a 25-μl reaction mixture prepared with appropriately diluted cDNAs, 0.2 μm 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 5′-RACE or R1, R2 and R3 for 3′-RACE with 1.0 μl of the 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 (5′-AAGCACCCTCAGACCACTTACTC-3′ and 5′-TTTGCAAGGCTCCTTCCCTTTCC-3′) based on the sequences obtained from the above cDNA fragments. PCR was carried out in a 25-μl reaction mixture prepared with appropriately diluted cDNAs, 0.2 μm primers, 1.25 units of KlenTaq DNA polymerase (CLONTECH) 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. CHO cells expressing human APJ (CHO-A10) were established as described previously (2.Tatemoto K. Hosoya M. Habata Y. Fujii R. Kakegawa T. Zou M.-X. Kawamata Y. Fukusumi S. Hinuma S. Kitada C. Kurokawa T. Onda H. Fujino M. Biochem. Biophys. Res. Commun. 1998; 251: 471-476Crossref PubMed Scopus (1289) Google Scholar). CHO cells expressing rat APJ were prepared principally by the same method. Poly(A)+ RNAs were prepared from the tissues of adult (8–12-week-old) and neonate (0–3-day-old) Wistar rats as described previously (9.Habata Y. Fujii R. Hosoya M. Fukusumi S. Kawamata Y. Hinuma S. Kitada C. Nishizawa N. Murosaki S. Kurokawa T. Onda H. Tatemoto K. Fujino M. Biochim. Biophys. Acta. 1999; 1452: 25-35Crossref PubMed Scopus (303) Google Scholar). We quantified rat APJ mRNA by means of a Prism 7700 sequence detector (PE Biosystems) with a primer set (5′-CCACCTGGTGAAGACTCTCTACA-3′ and 5′-TGACGTAACTGATGCAGGTGC-3′) and a probe labeled with fluorescent dyes (5′-(FAM)-TGACAGCTTCCTCATGAATGTCTTTCCC-(TAMRA)-3′). RT-PCR was carried out in a 25-μl reaction mixture prepared with a TaqMan PCR core reagent kit (PE Biosystems) containing an appropriately diluted cDNA solution, 0.2 μm each primer, and 0.1 μm probe. PCR was performed under the following conditions: at 50 °C for 10 min for the reaction of uracil-N-glycosylase 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 106 copies. Rat glyceraldehyde-3-phosphate dehydrogenase mRNA was also measured as an internal control as described previously (9.Habata Y. Fujii R. Hosoya M. Fukusumi S. Kawamata Y. Hinuma S. Kitada C. Nishizawa N. Murosaki S. Kurokawa T. Onda H. Tatemoto K. Fujino M. Biochim. Biophys. Acta. 1999; 1452: 25-35Crossref PubMed Scopus (303) Google Scholar). Extracellular acidification rates were measured with a Cytosensor (Molecular Devices Corp.) as described previously (2.Tatemoto K. Hosoya M. Habata Y. Fujii R. Kakegawa T. Zou M.-X. Kawamata Y. Fukusumi S. Hinuma S. Kitada C. Kurokawa T. Onda H. Fujino M. Biochem. Biophys. Res. Commun. 1998; 251: 471-476Crossref PubMed Scopus (1289) Google Scholar). CHO cells expressing human or rat APJ were dispersed with trypsin, and the cells were dispensed into cell capsules (Molecular Devices) at 2.7 × 105 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 μl/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 × 104 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. Methyl-isobutyl amirolide (MIA) (Research Biochemicals Inc., MA, USA) was dissolved in the low-buffered RPMI 1640 medium at 10 μm and exposed to the cells just before adding samples. Apelin-36 (human), [<Glu65]apelin-13, and [<Glu65,Nle75,Tyr77]apelin-13 were synthesized using an automatic peptide synthesizer (model 430, PE Biosystems) and purified by reversed-phase high performance liquid chromatography (reversed-phase HPLC) as described previously (2.Tatemoto K. Hosoya M. Habata Y. Fujii R. Kakegawa T. Zou M.-X. Kawamata Y. Fukusumi S. Hinuma S. Kitada C. Kurokawa T. Onda H. Fujino M. Biochem. Biophys. Res. Commun. 1998; 251: 471-476Crossref PubMed Scopus (1289) Google Scholar). [<Glu65,Nle75,Tyr77]apelin-13 was radioiodinated with Na125I (IMS-30, Amersham Pharmacia Biotech) by a method using lactoperoxidase (Sigma) as described elsewhere (10.Ohtaki T. Watanabe T. Ishibashi Y. Kitada C. Tsuda M. Gottschall P.E. Arimura A. Fujino M. Biochem. Biophys. Res. Commun. 1990; 171: 838-844Crossref PubMed Scopus (68) Google Scholar). After the reaction, the labeled and unlabeled peptides were separated by reversed-phase HPLC. Aliquots of the labeled peptide were stored at −30 °C until used. The membrane fractions of CHO-A10 cells prepared by the method as described previously (11.Fukusumi S. Kitada C. Takekawa S. Kizawa H. Sakamoto J. Miyamoto M. Hinuma S. Kitano K. Fujino M. Biochem. Biophys. Res. Commun. 1997; 232: 157-163Crossref PubMed Scopus (144) Google Scholar) were incubated with [125I][<Glu65,Nle75,Tyr77]apelin-13 in 100 μl 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 [<Glu65,Nle75,Tyr77]apelin-13 was simultaneously added to the wells. After incubation, bound and free radioactivities were separated through rapid filtration using the glass-fiber 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.Scatchard G. Ann. N. Y. Acad. Sci. 1949; 51: 660-672Crossref Scopus (17749) Google Scholar). CHO-A10 cells were seeded at 1 × 105 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 μm unlabeled [<Glu65,Nle75,Tyr77]apelin-13 was added to the wells. The cells were incubated with 200 pm[125I][<Glu65,Nle75,Tyr77]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[<Glu65]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 were performed with 96-well microchemotaxis chambers (Neuro Probe). [<Glu65]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 μl of each diluted solution was added to separate lower chambers. A polyvinylpyrrolidone-free polycarbonate-framed filter with 5-μm pores (Neuro Probe), after being precoated with 10 μg/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 × 105 cells/200 μl/well were added to the upper chamber. The chemotaxis chamber was incubated at 37 °C for 4 h in a CO2 incubator with 5% CO2 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). Colostrum from Holstein cows was boiled for 15 min, supplemented with up to 1m acetic acid, and homogenized using a Polytron homogenizer. The clear supernatant prepared by centrifugation was fractionated as described previously (2.Tatemoto K. Hosoya M. Habata Y. Fujii R. Kakegawa T. Zou M.-X. Kawamata Y. Fukusumi S. Hinuma S. Kitada C. Kurokawa T. Onda H. Fujino M. Biochem. Biophys. Res. Commun. 1998; 251: 471-476Crossref PubMed Scopus (1289) Google Scholar). In brief, the fraction eluted with 30% acetonitrile in C18 open column (Prep C18, 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 C18 column (Waters). After lyophilization, this fraction was applied on Superdex Peptide gel filtration column (Amersham Pharmacia Biotech) chromatography. Synthetic apelin-36 and [<Glu65]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.Habata Y. Fujii R. Hosoya M. Fukusumi S. Kawamata Y. Hinuma S. Kitada C. Nishizawa N. Murosaki S. Kurokawa T. Onda H. Tatemoto K. Fujino M. Biochim. Biophys. Acta. 1999; 1452: 25-35Crossref PubMed Scopus (303) Google Scholar). A rat APJ cDNA was isolated from rat brain poly(A)+ 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 [<Glu65]apelin-13 in a dose-dependent manner in the microphysiometric assay (data not shown). 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 × 105 to 9.1 × 105 copies/ng of poly(A)+ RNA except for pituitary, heart, and mammary gland (1.1 × 106 to 2.2 × 106 copies/ng of poly(A)+ RNA) and skeletal muscle (4.6 × 106 copies/ng of poly(A)+ RNA). We exposed CHO-A10 cells with [<Glu65]apelin-13 and human apelin-36 for a relatively long time (7 min 2 s). As shown in Fig.3, the promotion of acidification rate induced by [<Glu65]apelin-13 reached a maximum at 7 cycles under the experimental conditions employed here. The maximum acidification rate induced by [<Glu65]apelin-13 at 0.1 nm was 140%, and it reached a plateau (i.e.approximately 180%) at 1 and 10 nm. After the removal of [<Glu65]apelin-13, the elevated acidification rates gradually declined and returned to the basal level by 20 cycles at all doses examined. The acidification rate promoted by apelin-36 reached a maximum at the same number of cycles as [<Glu65]apelin-13. Although the maximum acidification rate induced by apelin-36 at 1 nm was lower (i.e. 120%) than that of [<Glu65]apelin-13, it reached the same plateau level as [<Glu65]apelin-13 at 10 and 100 nm. However, changes in the acidification rate after removal of the samples were quite distinctive between [<Glu65]apelin-13 and apelin-36; the elevated acidification rates induced by apelin-36 at 10 to 100 nmwere kept at a high level even after 20 cycles. In order to compare intracellular mechanisms by which [<Glu65]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.Tatemoto K. Hosoya M. Habata Y. Fujii R. Kakegawa T. Zou M.-X. Kawamata Y. Fukusumi S. Hinuma S. Kitada C. Kurokawa T. Onda H. Fujino M. Biochem. Biophys. Res. Commun. 1998; 251: 471-476Crossref PubMed Scopus (1289) Google Scholar, 9.Habata Y. Fujii R. Hosoya M. Fukusumi S. Kawamata Y. Hinuma S. Kitada C. Nishizawa N. Murosaki S. Kurokawa T. Onda H. Tatemoto K. Fujino M. Biochim. Biophys. Acta. 1999; 1452: 25-35Crossref PubMed Scopus (303) Google Scholar), APJ was thought to couple to the inhibitory G protein, Gi. We thus first tested the effects of PTX treatment. As shown in Fig.4, both [<Glu65]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 Gi. On the other hand, when we treated the CHO-A10 cells with MIA, the specific inhibitor for Na+/H+ exchanger (13.Neve K.A. Rosser M.P. Barber D.L. Methods Neurosci. 1995; 25: 225-241Crossref Scopus (5) Google Scholar), 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+/H+ 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.Figure 5Effects of MIA treatment on extracellular acidification rates. The running media were replaced by medium with (●) or without (○) 10 μm MIA, and the cells were incubated until stable acidification rates were obtained. Then, the cells were stimulated with 100 nm[<Glu65]apelin-13 (A) or apelin-36 (B) for 7 min 2 s, corresponding to cycles 4–7 as indicated by a boldface bar. The basal acidification rates in the first three cycles were normalized to 100%.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We radioiodinated an analogue of [<Glu65]apelin-13 (i.e.[<Glu65,Nle75,Tyr77]apelin-13) to use for receptor binding experiments. We used this analogue for the following reasons. As there was no tyrosine residue for radioiodination using lactoperoxidase in the sequence of [<Glu65]apelin-13, we substituted Phe77 with Tyr77. [<Glu65,Tyr77]apelin-13 was equivalently potent to the [<Glu65]apelin-13 in the microphysiometric assay (data not shown). In addition, [<Glu65,Nle75,Tyr77]apelin-13 was designed to prevent possible oxidization at Met75during the labeling reactions, as the activity of [<Glu65]apelin-13 was significantly decreased by oxidation (data not shown). The substitution of Met75 to norleucine (Nle75) did not reduce the agonistic activity. By the microphysiometric assay, we confirmed that the agonistic activity of [<Glu65,Nle75, Tyr77]apelin-13 remained after the iodination (data not shown). [125I][<Glu65,Nle75,Tyr77]apelin-13 specifically bound to intact CHO-A10 cells and their membrane preparations. Scatchard plot analyses for the binding of [125I][<Glu65,Nle75,Tyr77]apelin-13 to the membranes of CHO-A10 cells represented a single class of high affinity binding sites with a K d of 22.3 ± 2.7 pm and a B max of 3.01 ± 0.07 pmol/mg of protein (Fig. 6). In the competitive binding experiments, [<Glu65]apelin-13 and human apelin-36 effectively inhibited the binding of [125I][<Glu65,Nle75,Tyr77]apelin-13 to the CHO-A10 cell membranes, and their IC50 values were 1.4 ± 0.1 and 4.8 ± 0.24 nm, respectively (Fig. 7).Figure 7Competitive inhibition of radiolabeled apelin binding to APJ by [<Glu65]apelin-13 and apelin-36.[125I][<Glu65,Nle75,Tyr77]apelin-13 (100 pm) binding to the membrane preparations of CHO-A10 cells (0.25 μg/100 μl) was examined in the presence of [<Glu65]apelin-13 (○) and apelin-36 (●) at the indicated concentrations. The bound and free ligands were separated after incubation for 90 min at room temperature. The amounts of nonspecific binding were estimated by adding 1 μmunlabeled [<Glu65,Nle75,Tyr77]apelin-13 to the reaction buffer. Each symbol represents the mean of triplicate determinations. Standard error bars are invisible because they lie inside in the symbols.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In order to assess the dissociation of [<Glu65]apelin-13 and apelin-36 to APJ, we compared the binding of [125I][<Glu65, Nle75,Tyr77]apelin-13 to CHO-A10 cells pretreated with [<Glu65]apelin-13 or apelin-36. At first, we examined the time course of the association of [125I][<Glu65,Nle75,Tyr77]apelin-13 on intact CHO-A10 cells and found that the labeled ligand rapidly bound to the cells within 30 min (Fig.8 A). We therefore exposed CHO-A10 cells to the excess amount of each peptide at a concentration of 1 mm for 90 min, washed out unbound peptides, and then determined the amount of [125I][<Glu65,Nle75,Tyr77]apelin-13 binding to the CHO-A10 cells. The radiolabeled ligand effectively bound to the cells even after pretreatment with a high concentration of [<Glu65]apelin-13 (Fig. 8 B). The time-course kinetics of [125I][<Glu65,Nle75,Tyr77]apelin-13 binding to the cells pretreated with [<Glu65]apelin-13 was close to that of untreated cells. In contrast, the binding of the radiolabeled ligand to CHO-A10 cells treated with apelin-36 was very low, suggesting that apelin-36 bound to APJ cannot be easily replaced with [125I][<Glu65,Nle75,Tyr77]apelin-13. As a functional characterization of APJ, we examined the chemotactic action of apelin. As shown in Fig.9, [<Glu65]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 [<Glu65]apelin-13. The dose-response curves for [<Glu65]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. A peptide-enriched fraction was prepared from bovine colostrum by a combination of C18 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 [<Glu65]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 [<Glu65]apelin-13, respectively, are produced at least in bovine colostrum, although the long forms of apelin appeared to be dominant. 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.Tatemoto K. Hosoya M. Habata Y. Fujii R. Kakegawa T. Zou M.-X. Kawamata Y. Fukusumi S. Hinuma S. Kitada C. Kurokawa T. Onda H. Fujino M. Biochem. Biophys. Res. Commun. 1998; 251: 471-476Crossref PubMed Scopus (1289) Google Scholar, 9.Habata Y. Fujii R. Hosoya M. Fukusumi S. Kawamata Y. Hinuma S. Kitada C. Nishizawa N. Murosaki S. Kurokawa T. Onda H. Tatemoto K. Fujino M. Biochim. Biophys. Acta. 1999; 1452: 25-35Crossref PubMed Scopus (303) Google Scholar), 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.Habata Y. Fujii R. Hosoya M. Fukusumi S. Kawamata Y. Hinuma S. Kitada C. Nishizawa N. Murosaki S. Kurokawa T. Onda H. Tatemoto K. Fujino M. Biochim. Biophys. Acta. 1999; 1452: 25-35Crossref PubMed Scopus (303) Google Scholar). 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.Edinger A.L. Hoffman T.L. Sharron M. Lee B. Yi Y. Choe W. Kolson D.L. Mitrovic B. Zhou Y. Faulds D. Collman R.D. Hesselgesser J. Horuk R. Doms R.W. J. Virol. 1998; 72: 7934-7940Crossref PubMed Google Scholar). 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, 2M. Hosoya, Y. Kawamata, S. Fukusumi, R. Fujii, Y. Habata, S. Hinuma, C. Kitada, S. Honda, T. Kurokawa, H. Onda, O. Nishimura, and M. Fujino, unpublished data. 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.Tatemoto K. Hosoya M. Habata Y. Fujii R. Kakegawa T. Zou M.-X. Kawamata Y. Fukusumi S. Hinuma S. Kitada C. Kurokawa T. Onda H. Fujino M. Biochem. Biophys. Res. Commun. 1998; 251: 471-476Crossref PubMed Scopus (1289) Google Scholar, 9.Habata Y. Fujii R. Hosoya M. Fukusumi S. Kawamata Y. Hinuma S. Kitada C. Nishizawa N. Murosaki S. Kurokawa T. Onda H. Tatemoto K. Fujino M. Biochim. Biophys. Acta. 1999; 1452: 25-35Crossref PubMed Scopus (303) Google Scholar). Based on the analyses for the endogenous apelin purified from the bovine stomach, a mature peptide (apelin-36) has been found to consist of 36 amino acid residues, starting from Leu42 and running to the C terminus of Phe77. 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, [<Glu65]apelin-13, and apelin-17 showed stronger extracellular acidifying activities than apelin-36 to CHO-A10 cells in dose-response analyses. We synthesized [<Glu65]apelin-13 as a pyroglutamylated form of apelin-13, and the two peptides were equipotent in the acidification rate promoting activity (2.Tatemoto K. Hosoya M. Habata Y. Fujii R. Kakegawa T. Zou M.-X. Kawamata Y. Fukusumi S. Hinuma S. Kitada C. Kurokawa T. Onda H. Fujino M. Biochem. Biophys. Res. Commun. 1998; 251: 471-476Crossref PubMed Scopus (1289) Google Scholar). Because [<Glu65]apelin-13 is structurally more stable than apelin-13, we have mainly used [<Glu65]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 [<Glu65]apelin-13 and apelin-36. Somatostatin exists in multiple forms (i.e. somatostatin-14, somatostatin-25, and somatostatin-28) (15.Bohlen P. Brazeau P. Benoit R. Ling N. Esch F. Guillemin R. Biochem. Biophys. Res. Commun. 1980; 96: 725-734Crossref PubMed Scopus (55) Google Scholar). 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.Habata Y. Fujii R. Hosoya M. Fukusumi S. Kawamata Y. Hinuma S. Kitada C. Nishizawa N. Murosaki S. Kurokawa T. Onda H. Tatemoto K. Fujino M. Biochim. Biophys. Acta. 1999; 1452: 25-35Crossref PubMed Scopus (303) Google Scholar). 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 Leu42, Gly47, and Ser50 (which would correspond to apelin-36, -31, and -28), exist in bovine milk (9.Habata Y. Fujii R. Hosoya M. Fukusumi S. Kawamata Y. Hinuma S. Kitada C. Nishizawa N. Murosaki S. Kurokawa T. Onda H. Tatemoto K. Fujino M. Biochim. Biophys. Acta. 1999; 1452: 25-35Crossref PubMed Scopus (303) Google Scholar). In this study, we analyzed whether short forms of apelin corresponding to [<Glu65]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 [<Glu65]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.Habata Y. Fujii R. Hosoya M. Fukusumi S. Kawamata Y. Hinuma S. Kitada C. Nishizawa N. Murosaki S. Kurokawa T. Onda H. Tatemoto K. Fujino M. Biochim. Biophys. Acta. 1999; 1452: 25-35Crossref PubMed Scopus (303) Google Scholar). We therefore consider that the short forms as well as long forms of apelin are produced and function under some physiological conditions. In our receptor binding assays, [125I][<Glu65,Nle75,Tyr77]apelin-13 specifically bound to CHO-A10 cells with a high affinity, and [<Glu65]apelin-13 was more efficient than apelin-36 in the competitive analyses. In the dose-response relationships in the microphysiometric assay, [<Glu65]apelin-13 was proven to be much more potent than apelin-36 (2.Tatemoto K. Hosoya M. Habata Y. Fujii R. Kakegawa T. Zou M.-X. Kawamata Y. Fukusumi S. Hinuma S. Kitada C. Kurokawa T. Onda H. Fujino M. Biochem. Biophys. Res. Commun. 1998; 251: 471-476Crossref PubMed Scopus (1289) Google Scholar). These results unequivocally demonstrated that [<Glu65]apelin-13 could more efficiently associate with APJ than apelin-36. On the other hand, the pretreatment of CHO-A10 cells with [<Glu65]apelin-13 did not efficiently suppress the binding of [125I][<Glu65,Nle75,Tyr77]apelin-13. [<Glu65]apelin-13 was thus supposed to rapidly dissociate from APJ so that the labeled ligand could replace with [<Glu65]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 [<Glu65]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, [<Glu65]apelin-13 was more potent than apelin-36. As we have demonstrated previously, [<Glu65]apelin-13 also shows higher activity than apelin-36 in the cAMP production-inhibitory assay using CHO-A10 cells (9.Habata Y. Fujii R. Hosoya M. Fukusumi S. Kawamata Y. Hinuma S. Kitada C. Nishizawa N. Murosaki S. Kurokawa T. Onda H. Tatemoto K. Fujino M. Biochim. Biophys. Acta. 1999; 1452: 25-35Crossref PubMed Scopus (303) Google Scholar). 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 vivosituation, if APJ is transiently exposed to an apelin peptide, apelin-36 might show greater activity than [<Glu65]apelin-13. In any case, further studies are necessary 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. We thank Drs. Yasuhiro Sumino, Yoshihiro Ishibashi, and Takuya Watanabe and Yoshio Matsumoto for their helpful advice and collaboration.