Modification of the Terminal Residue of Apelin-13 Antagonizes Its Hypotensive Action
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内分泌学杂志 2005年第1期
Departments of Pharmacology (D.K.L., V.R.S., S.R.G., B.F.O.) and Medicine (S.R.G.), University of Toronto, Toronto, Ontario, Canada M5S 1A8; and The Centre for Addiction and Mental Health (T.N., R.C., S.R.G., B.F.O.), Toronto, Ontario, Canada M5S 2S1
Address all correspondence and requests for reprints to: Brian F. O’Dowd, Department of Pharmacology, University of Toronto, Medical Science Building, 1 King’s College Circle, Room 4352, Toronto, Ontario, Canada M5S 1A8. E-mail: brian.odowd@utoronto.ca.
Abstract
The apelin peptide is the endogenous ligand for the apelin G protein-coupled receptor. The distribution of the apelin peptides and receptor are widespread in the central nervous system and periphery, with reported roles in the hypothalamic-pituitary-adrenal axis, blood pressure regulation and as one of the most potent positive inotropic substances yet identified. In this report, we show that in native tissues preproapelin exists as a dimer. Dimeric preproapelin was reduced to monomers by dithiothreitol treatment, indicating disulfide linkages. To evaluate the role of the carboxyl-terminal phenylalanine in the hypotensive action of apelin-13, analogs were generated and tested for their role on blood pressure regulation. Injections of apelin-13 and apelin-12 (15 μg/kg) into spontaneously hypertensive rats lowered systolic and diastolic blood pressure to result in decreases of approximately 60% and 15% in mean arterial blood pressure, respectively. Apelin-13(13[D-Phe]) treatment did not differ from apelin-13 in either efficacy or duration of effect, whereas apelin-13(F13A) revealed a loss of function. However, concomitant administration of apelin-13(F13A) (30 μg/kg) blocked hypotensive effects of apelin-13 (15 μg/kg), which revealed that apelin-13(F13A) behaved as an apelin-specific antagonist.
Introduction
THE LARGE NUMBER of identified G protein-coupled receptor (GPCR) genes has resulted in the ongoing characterization of novel receptor and ligand systems, in areas such as physiology, anatomic distribution, and tissue-specific functions (1). One such GPCR, the apelin receptor, was originally discovered in our laboratory and had 40–50% shared identity with the angiotensin AT1 receptor in the transmembrane regions (2). Two major isoforms of a peptide, apelin-36 and apelin-13, were identified as the endogenous ligands for this receptor (3), which also shared some structural similarities to the angiotensin II peptide (4). Thus, for this novel peptidergic system, there is much interest in the elucidation of the pharmacology and endocrinology of the apelin receptor system.
The apelinergic system has a widespread distribution in both the central nervous system (CNS) and periphery, particularly in the heart, kidney, lung, and mammary gland (4, 5, 6, 7, 8). In the CNS, the distribution of apelin and its receptor in the hippocampus and the hypothalamic supraoptic and paraventricular nuclei led to the documentation of apelin-induced reduction of vasopressin release, increases in plasma ACTH and corticosterone, as well as modulation of drinking behavior and food intake (4, 9, 10, 11, 12). These effects were indicative of a role for apelin in the hypothalamic-pituitary-adrenal axis, which was further confirmed by increased levels of apelin receptor mRNA resulting from acute and chronic restraint stress and after adrenalectomy (13). In addition to these physiological effects, the apelinergic system may possess novel cellular mechanisms. We recently observed the apelin receptor localized in cell nuclei in the cerebellum and paraventricular nuclei of the hypothalamus, which suggests novel mechanisms of apelin signal transduction in the brain (14).
Physiological effects of apelin have also been studied in the periphery, with apelin shown to lower blood pressure after iv injection into rats (4, 15, 16). This effect was abolished in the presence of a nitric oxide synthase inhibitor, suggesting that apelin lowers blood pressure through a nitric oxide mechanism (15). Furthermore, the restricted localization of apelin within endothelial cells of blood vessels suggests a role as a paracrine mediator of blood vessel contractility (17). Blood pressure regulation by apelin also appears to be modulated by the CNS. In the rat brainstem, microinjections of apelin-13 into the nucleus tractus solitarius and the rostral ventrolateral medulla modified phrenic nerve activity and blood pressure, suggesting not only a role in controlling arterial pressure but respiration as well (18). Apelin has also been observed to mediate effects directly on the heart. In fact, apelin is one of the most potent endogenous positive inotropic substances, enhancing cardiac contractility (19). Recently, low apelin peptide and receptor mRNA levels were found in failing human hearts, which were up-regulated after the addition of a left ventricular assist device (20).
We now report that preproapelin exists in a dimeric form in native tissues and demonstrate the enhanced effects of apelin-13 in lowering blood pressure in spontaneously hypertensive rats (SHR), compared with normal Wistar rats. Recently a novel angiotensin-converting enzyme, ACE2, was discovered to hydrolyze apelin-13 and apelin-36 (21). In addition, substitution of the carboxyl-terminal phenylalanine has been shown to decrease binding affinity and even abolish the activity of apelin-13 in vitro (22, 23). Intriguingly, substitution of this homologous phenylalanine in angiotensin II has resulted in the generation of angiotensin II antagonists. In this report we disclose a novel antagonist for the apelin system, apelin-13(F13A), which abolished apelin-13 blood pressure-lowering effects in vivo in hypertensive animals. Together, these findings suggest that the carboxyl-terminal phenylalanine plays a critical role in apelin function.
Materials and Methods
Cell culture and transfection of cells
COS-7 monkey kidney cells (American Type Culture Collection, Manassas, VA) were maintained as monolayer cultures at 37 C in MEM supplemented with 10% fetal bovine serum and antibiotics. DNA encoding the rat preproapelin peptide was obtained as previously described (4). Cells were transiently transfected with cDNA by the use of Lipofectamine reagent (Life Technologies, Rockville, MD).
Generation of preproapelin transgenic mice
Briefly, rat preproapelin-encoding cDNA was inserted in the PcAGGS vector at the XhoI site. The vector’s backbone was subsequently removed by restriction enzyme digestion with StuI and ScaI. The remaining construct (550 bp) was purified and injected into mouse egg donor (strain C57BL/6xSJL) with multicopy insertion. Six litters were born (88 mice), of which 64 mice remained after 3 wk. Genotype analysis revealed 10 PcAGGS-positive mice. These mice were crossed with wild-type mice, and their litters were genotyped for PcAGGS vector.
Membrane preparation
All cells and tissues were washed extensively with PBS. Whole-cell lysates of COS-7 cells were prepared by polytron disruption in ice-cold 5:2 lysis buffer (containing 5 mM Tris-HCl, 2 mM EDTA buffer containing 5 μg/ml leupeptin, 10 μg/ml benzamidine, and 5 μg/ml soybean trypsin inhibitor). For mouse tissues, adult mice (wild-type and preproapelin transgenic) were killed and brain and peripheral tissues were removed and frozen on crushed dry ice. These samples were homogenized in ice-cold 5:2 lysis buffer using a tight pestle tissue grinder and were then centrifuged and disrupted using a sonicator. Protein levels were determined by the Bradford assay according to the manufacturer’s instructions (Bio-Rad, Hercules, CA).
Immunoblot analyses
Whole-cell and tissue lysates were treated with deoxyribonuclease and ribonuclease (0.5 μg/μl each; Marligen Biosciences, Inc., Ijamsville, MD) for 20 min at 37 C. Samples subjected to reducing conditions were treated with 50 mM dithiothreitol (DTT) and 100 mM iodoacetamide for 30 min at 60 C. Twenty-five micrograms of protein of lysates were solubilized in sample buffer consisting of 50 mM Tris-HCl (pH 6.5), 1% sodium dodecyl sulfate, 10% glycerol, 0.003% bromophenol blue, and 10% 2-mercaptoethanol. The samples were subjected to polyacrylamide gel electrophoresis with 10–20% acrylamide gels and electroblotted onto nitrocellulose as previously described (24). Apelin-36 immunoreactivity was revealed with a rabbit polyclonal antibody (catalog number G-057-15; Phoenix Pharmaceuticals, Inc., Belmont, CA) at a concentration of 500 ng/ml.
Blood pressure and heart rate bioassay
Animal experiments and care were approved by the University of Toronto Animal Care Committee. Male Wistar rats and SHR weighing 280–330 g and at approximately 15 wk of age (Charles River Laboratories, Wilmington, MA) were anesthetized with Inactin (Promonta, Hamburg, Germany) administered ip at 100 mg/kg body weight (B.W.). The Inactin solution was prepared fresh every experimental day at a concentration of 50 mg/ml saline (0.9% NaCl) and kept on ice. Rats were placed on a heating pad to maintain normal body temperature throughout the experimental procedure. Right carotid artery and left femoral vein were isolated by blunt dissection and cannulated with PE-50 polyethylene tubing and tied firmly in place. Blood pressure and heart rate were recorded via cannula inserted in the right carotid artery. The basal blood pressure for SHR was stabilized at 220/170 mm Hg systolic/diastolic blood pressure. Apelin peptides were injected in via PE-50 cannula fitted with a 23-gauge needle placed in the left femoral vein. Blood pressure was recorded using a pressure transducer connected to a physiograph (MK-III-S; Narco Bio-Systems, Austin, TX). The pressure recording system was calibrated each experimental day.
Injection procedure.
Each rat received iv injection of angiotensin II (30 ng/kg B.W.; A-9225; Sigma, St. Louis, MO) to determine responsiveness. Only those responding with an increase of 15–20 mm Hg systolic were used for the bioassay of apelin peptides (4). Rats received an equivolume of saline as control, and blood pressure and heart rate was monitored for 10 min before injection of apelin peptides. The apelin analogs tested included: apelin-13 (QRPRLSHKGPMPF), apelin-12 (RPRLSHKGPMPF), apelin-13(F13A) (QRPRLSHKGPMPA), apelin-13(13[D-Phe]) (QRPRLSHKGPMP[D-Phe]), and apelin-13(F13[D-Ala]) (QRPRLSHKGPMP[D-Ala]) (Advanced Protein Technology Centre, University of Toronto). The peptides were dissolved as a stock solution of 100 μg/ml in 0.9% saline and administered at 6–25 μg/kg B.W. The blood pressure responses to iv injections of apelin peptides were expressed as mean ± SE and reported as systolic/diastolic pressure, mean arterial pressure, and changes in heart rate. Statistical analyses between treatments were calculated by unpaired two-tailed Student’s t tests using GraphPad (San Diego, CA) software, with a value of P < 0.05 considered statistically significant.
Results
Detection of preproapelin
An apelin-36 antibody was used to detect apelin-immunoreactivity in tissues from wild-type mice, preproapelin transgenic mice, and in COS-7 cells expressing preproapelin. Apelin immunoreactivity was detected as a band of approximately 16 kDa in lysates of heart tissue obtained from wild-type mice (Fig. 1A, lane 2). This band was also observed in lysates of several brain regions, lung, kidney, liver, and spleen. Apelin immunoreactivity was detected as two bands at approximately 8 and 16 kDa in the transgenic mice (Fig. 1A, lane 3) and COS-7 cells (Fig. 1A, lane 1), which correlated with presumed monomeric and dimeric forms of preproapelin. Lysates from COS-7 cells were reduced using DTT treatment, which revealed only the 8-kDa band (Fig. 1B, lane 2). The absence of the 16-kDa preproapelin species after reduction by DTT indicated the preproapelin dimer is stabilized by disulfide bridges linking cysteine residues. Because only the dimeric form was observed in normal mice, these findings suggest preproapelin exists physiologically as a dimer. However, the absence of cysteine residues in mature apelin-36 and apelin-13 suggest these processed peptides are monomeric.
FIG. 1. Apelin-36 antibody immunoblot analyses. Immunoblot analysis of whole-cell lysates using the apelin-36 antibody. A, Shown are samples from COS-7 cells expressing rat preproapelin (lane 1) and heart tissue samples for a control mouse (lane 2) and a preproapelin-transgenic littermate (lane 3). B, Samples from COS-7 cells expressing rat preproapelin either untreated (lane 1) or treated with 50 mM DTT and 100 mM iodoacetamide for 30 min at 60 C (lane 2). For all lanes, 25 μg of protein was used. The immunoblots shown are representative of two independent experiments.
Blood pressure modulation by apelin analogs in rats
We were the first to report a hypotensive action of apelin-13 in normotensive rats (4). In this study, we examined the effects of apelin and apelin analogs in a hypertensive rat model. Recognition of the critical nature of the carboxyl-terminal phenylalanine for apelin activity in vitro (22, 23) and as the site of ACE2 cleavage (21) prompted our investigation into the role of this residue on apelin function in vivo. Because apelin was previously shown to have a very short-lived effect in lowering blood pressure, we hypothesized that substituting a D-isomer amino acid in the carboxyl-terminal position would inhibit ACE2-mediated cleavage of the apelin peptide (Table 1). We also determined whether an alanine substitution in the carboxyl terminus of apelin-13 would yield an apelin-specific antagonist, as was previously shown for angiotensin II and saralasin (Table 1B), an early angiotensin II antagonist (25).
TABLE 1. Sequence of apelin analogs and related peptides
We compared the hypotensive effects of apelin in SHR and Wistar rats. The basal resting blood pressure values for SHR and Wistar rats were approximately 220/170 and 140/110 mm Hg respectively. Overall, administration of both apelin-12 and apelin-13 resulted in immediate decreases of both systolic and diastolic blood pressure with maximal responses within 1 min of injection. Injections of apelin-12 iv in doses of 6, 9, and 15 μg/kg B.W. revealed that only the 15-μg/kg dose induced a decrease in mean arterial blood pressure (MABP). Apelin-13 (15 μg/kg) administered iv to SHR and Wistar rats resulted in approximately 60 and 30% reduction in MABP, respectively (Fig 2A). In contrast to this, 15 μg/kg apelin-12 revealed approximately 15 and 30% decreases in MABP in SHR and Wistar rats, respectively. Because the magnitude of effect was greatest for apelin-13 in the SHR (from a basal blood pressure of 217/167 to 128/93 mm Hg within 1 min after injection), we used this rat model to test various analogs of the apelin-13 peptide. Injection of 15 μg/kg of apelin-13(13[D-Phe]) induced a rapid decrease of MABP, similar to apelin-13. However, the duration of effect was similar to apelin-13, suggesting that the D-isomer conformation of the phenylalanine did not affect efficacy or time course of action. Administration of 15 μg/kg of apelin-13(F13A) and apelin-13(F13[D-Ala]) revealed no significant reduction of MABP from baseline (Fig. 2B), indicating a loss of apelin effect. Overall, apelin-13 and apelin-13(13[D-Phe]) were the most potent hypotensive agents in SHR, whereas apelin-13(F13A) and apelin-13(F13[D-Ala]) produced no significant changes in blood pressure (summarized in Fig. 2C). For apelin and all apelin analogs, iv administration did not induce a significant change in heart rate, as illustrated by a representative trace for apelin-13 (Fig. 2A).
FIG. 2. Effects of apelin peptides on MABP in anesthetized rats. A, Time-course changes in MABP (% basal) after iv administration of 15 μg/kg apelin-12 or apelin-13 into Wistar or SHR at time 0 (arrow). Included is a representative time-course in heart rate (% basal) after iv administration of 15 μg/kg apelin-13 into SHR (open circles), as plotted vs. the right y-axis (Heart Rate). Data are mean ± SEM. B, Time-course changes in MABP (% basal) after iv administration of 15 μg/kg apelin-13 analogs into SHR at time 0 (arrow). Data are mean ± SEM. C, Comparison of the maximum decrease in MABP (% basal) of the apelin analogs in SHR. *, P < 0.0001 vs. apelin-13(F13A) treatment.
Whereas apelin-13(F13A) did not alter blood pressure on its own, we speculated that a concomitant dose may antagonize the hypotensive effects of apelin-13, as shown for saralasin and angiotensin II. SHR responsive to 15 μg/kg of apelin-13 were allowed to recover for 40 min, after which a combined dose of 15 μg/kg apelin-13 and 30 μg/kg apelin-13(F13A) showed a complete blockade of apelin hypotensive action (Fig. 3A). To ensure this was not the result of desensitization to apelin-13, 15 μg/kg of apelin-13 was injected after another 40-min recovery period, which resulted in a similar decrease in blood pressure as the first apelin-13 injection. This antagonism by apelin-13(F13A) was dose dependent, as shown by the effects of increasing doses of 0.1, 3, 10, and 30 μg/kg of apelin-13(F13A) administered concomitantly with 15 μg/kg apelin-13 (Fig. 3B). Furthermore, antagonism by apelin-13(F13A) was observed as specific against apelin-13, because no significant difference was observed between the increases in MABP of SHR treated with 40 ng/kg angiotensin II with or without coinjection of 30 μg/kg apelin-13(F13A) (Fig. 3C).
FIG. 3. Apelin-13(F13A) is an antagonist for apelin-13 in vivo. A, Time-course changes in systolic (open squares) and diastolic (open triangles) blood pressure (mm Hg) after iv administration of 15 μg/kg apelin-13 (closed arrow) and 30 μg/kg apelin-13(F13A) (open arrow) into SHR. Data are mean ± SEM (n = 3). B, Dose response of apelin-13(F13A) iv administration at 0.1 (n = 3), 3 (n = 4), 10 (n = 5), and 30 (n = 5) μg/kg with a concomitant injection of apelin-13 (15 μg/kg) into SHR. Shown is the MABP (% basal) at maximum decrease within 1 min after injection. Data are mean ± SEM. C, Comparison of the increase in MABP (% over basal) after iv administration of angiotensin II (40 ng/kg) alone or angiotensin II (40 ng/kg) and apelin-13(F13A) (30 μg/kg) together. Data are mean ± SEM (n = 5) with no significant difference observed between the two treatments.
Discussion
In this report, we demonstrate that apelin can lower blood pressure in normotensive rats and to a much greater extent in the hypertensive SHR. The similar decreases in both systolic and diastolic blood pressure were indicative of an arterial vasodilation effect. Decrements in blood pressure were dramatically attenuated by coadministration of the apelin-13 analog with a single amino acid substitution of the carboxyl-terminal phenylalanine, yielding the successful development of a novel apelin antagonist. In addition, we observed preproapelin to be exclusively dimeric in vivo.
To compare the hypotensive effects of apelin in normotensive and hypertensive rats, we tested the effects of iv injections of apelin in Wistar rats and SHR. We observed no differences in the hypotensive effects between 15-μg/kg rat B.W. doses of apelin-12 and apelin-13 in Wistar rats. Another group reported apelin-12 as more potent than apelin-13 in reducing MABP in Wistar rats (15). These disparate results may perhaps be explained by the different anesthetic treatments used on these rats. Apelin-13 had a much greater potency than apelin-12 in SHR, decreasing MABP by approximately 60% compared with approximately 15% (i.e. respective reductions of 90 and 15 mm Hg). Due to the high sensitivity observed in SHR with apelin-13, we performed subsequent experiments using SHR to test the apelin-13 analogs.
As a basis for designing potential apelin antagonists, we examined an earlier successful strategy used for the closely related angiotensin II peptide. Before the development of angiotensin receptor subtype-specific antagonists and ACE inhibitors, an angiotensin II analog, saralasin (angiotensin II[Sar1, Val5, Ala8]) was found to act as an antagonist to angiotensin II pressor effects in rats (25). We hypothesized that a similar substitution of alanine in place of the carboxyl-terminal phenylalanine in apelin-13 would produce an effective antagonist. The dramatic reduction of blood pressure by apelin-13 was completely attenuated in a second injection combined with apelin-13(F13A). This effect was not due to apelin receptor desensitization, since a subsequent administration of apelin-13 displayed an identical reduction in blood pressure, indicating intact receptor sensitivity. Apelin-13(F13A) antagonism of apelin-13 was observed to be dose-dependent and did not affect the activity of angiotensin II on blood pressure, which suggests specific antagonism at the apelin receptor. Furthermore, the parallel changes in both systolic and diastolic blood pressure were evidence of both arterial and venous dilator effects, which corresponded with a previous study which described apelin-induced decreases in both MABP and mean circulatory filling pressure (16). The apelin-13(F13A) analog has been examined with varying results in vitro, with regard to ligand binding affinities and effect on second messenger systems (22, 23, 26). These in vitro differences have not yet been reconciled, whereas we observed a clear antagonist action in vivo.
Recently, a second angiotensin-converting enzyme-related carboxypeptidase (ACE2) was discovered to hydrolyze both angiotensin II and apelin, which resulted in cleavage of the carboxyl-terminal phenylalanine residues from both peptides. We hypothesized the inclusion a D-isomer carboxyl amino acid residue would prevent hydrolysis of apelin-13 and perhaps heighten or extend the hypotensive effects in vivo. However, little difference was observed between apelin-13 and apelin-13(13[D-Phe]) peptides, suggesting that the D-conformation of the carboxyl-terminal phenylalanine was irrelevant to the hydrolysis activity of ACE2.
In wild-type mice, preproapelin was detected as a dimeric protein. Monomeric preproapelin was only detected in cells and transgenic mice overexpressing preproapelin, suggesting that preproapelin exists exclusively in dimeric form in vivo. Reduction of dimeric to monomeric preproapelin indicated the dimerization was a consequence of disulfide bridges formed between cysteine residues. Sequence analysis revealed two cysteines flanking the signal sequence at the amino-terminal end of human and bovine preproapelin. These cysteines are conserved in rat preproapelin with an additional cysteine embedded within the signal sequence. However, no cysteines were observed in the processed apelin-36 and apelin-13 peptides, which are likely monomeric. Dimerization has been revealed as a prerequisite step for proper processing of other prepropeptides including prosomatostatin-II (27), prouroguanylin (28), and the human nerve growth factor (29).
In conclusion, we report preproapelin is dimeric in native tissues and in transfected cells. Functionally we demonstrate a pronounced hypotensive effect of apelin-13 in a hypertensive rate model compared with normotensive rats. We have developed a novel apelin receptor antagonist generated by an alanine substitution in the carboxyl-terminal end of apelin-13. Because there are no known selective antagonists for the apelinergic system, the identification of a highly selective apelin antagonist should greatly aid in the characterization of this receptor system and possibly lead to novel advances in apelin-related therapeutic treatment.
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Address all correspondence and requests for reprints to: Brian F. O’Dowd, Department of Pharmacology, University of Toronto, Medical Science Building, 1 King’s College Circle, Room 4352, Toronto, Ontario, Canada M5S 1A8. E-mail: brian.odowd@utoronto.ca.
Abstract
The apelin peptide is the endogenous ligand for the apelin G protein-coupled receptor. The distribution of the apelin peptides and receptor are widespread in the central nervous system and periphery, with reported roles in the hypothalamic-pituitary-adrenal axis, blood pressure regulation and as one of the most potent positive inotropic substances yet identified. In this report, we show that in native tissues preproapelin exists as a dimer. Dimeric preproapelin was reduced to monomers by dithiothreitol treatment, indicating disulfide linkages. To evaluate the role of the carboxyl-terminal phenylalanine in the hypotensive action of apelin-13, analogs were generated and tested for their role on blood pressure regulation. Injections of apelin-13 and apelin-12 (15 μg/kg) into spontaneously hypertensive rats lowered systolic and diastolic blood pressure to result in decreases of approximately 60% and 15% in mean arterial blood pressure, respectively. Apelin-13(13[D-Phe]) treatment did not differ from apelin-13 in either efficacy or duration of effect, whereas apelin-13(F13A) revealed a loss of function. However, concomitant administration of apelin-13(F13A) (30 μg/kg) blocked hypotensive effects of apelin-13 (15 μg/kg), which revealed that apelin-13(F13A) behaved as an apelin-specific antagonist.
Introduction
THE LARGE NUMBER of identified G protein-coupled receptor (GPCR) genes has resulted in the ongoing characterization of novel receptor and ligand systems, in areas such as physiology, anatomic distribution, and tissue-specific functions (1). One such GPCR, the apelin receptor, was originally discovered in our laboratory and had 40–50% shared identity with the angiotensin AT1 receptor in the transmembrane regions (2). Two major isoforms of a peptide, apelin-36 and apelin-13, were identified as the endogenous ligands for this receptor (3), which also shared some structural similarities to the angiotensin II peptide (4). Thus, for this novel peptidergic system, there is much interest in the elucidation of the pharmacology and endocrinology of the apelin receptor system.
The apelinergic system has a widespread distribution in both the central nervous system (CNS) and periphery, particularly in the heart, kidney, lung, and mammary gland (4, 5, 6, 7, 8). In the CNS, the distribution of apelin and its receptor in the hippocampus and the hypothalamic supraoptic and paraventricular nuclei led to the documentation of apelin-induced reduction of vasopressin release, increases in plasma ACTH and corticosterone, as well as modulation of drinking behavior and food intake (4, 9, 10, 11, 12). These effects were indicative of a role for apelin in the hypothalamic-pituitary-adrenal axis, which was further confirmed by increased levels of apelin receptor mRNA resulting from acute and chronic restraint stress and after adrenalectomy (13). In addition to these physiological effects, the apelinergic system may possess novel cellular mechanisms. We recently observed the apelin receptor localized in cell nuclei in the cerebellum and paraventricular nuclei of the hypothalamus, which suggests novel mechanisms of apelin signal transduction in the brain (14).
Physiological effects of apelin have also been studied in the periphery, with apelin shown to lower blood pressure after iv injection into rats (4, 15, 16). This effect was abolished in the presence of a nitric oxide synthase inhibitor, suggesting that apelin lowers blood pressure through a nitric oxide mechanism (15). Furthermore, the restricted localization of apelin within endothelial cells of blood vessels suggests a role as a paracrine mediator of blood vessel contractility (17). Blood pressure regulation by apelin also appears to be modulated by the CNS. In the rat brainstem, microinjections of apelin-13 into the nucleus tractus solitarius and the rostral ventrolateral medulla modified phrenic nerve activity and blood pressure, suggesting not only a role in controlling arterial pressure but respiration as well (18). Apelin has also been observed to mediate effects directly on the heart. In fact, apelin is one of the most potent endogenous positive inotropic substances, enhancing cardiac contractility (19). Recently, low apelin peptide and receptor mRNA levels were found in failing human hearts, which were up-regulated after the addition of a left ventricular assist device (20).
We now report that preproapelin exists in a dimeric form in native tissues and demonstrate the enhanced effects of apelin-13 in lowering blood pressure in spontaneously hypertensive rats (SHR), compared with normal Wistar rats. Recently a novel angiotensin-converting enzyme, ACE2, was discovered to hydrolyze apelin-13 and apelin-36 (21). In addition, substitution of the carboxyl-terminal phenylalanine has been shown to decrease binding affinity and even abolish the activity of apelin-13 in vitro (22, 23). Intriguingly, substitution of this homologous phenylalanine in angiotensin II has resulted in the generation of angiotensin II antagonists. In this report we disclose a novel antagonist for the apelin system, apelin-13(F13A), which abolished apelin-13 blood pressure-lowering effects in vivo in hypertensive animals. Together, these findings suggest that the carboxyl-terminal phenylalanine plays a critical role in apelin function.
Materials and Methods
Cell culture and transfection of cells
COS-7 monkey kidney cells (American Type Culture Collection, Manassas, VA) were maintained as monolayer cultures at 37 C in MEM supplemented with 10% fetal bovine serum and antibiotics. DNA encoding the rat preproapelin peptide was obtained as previously described (4). Cells were transiently transfected with cDNA by the use of Lipofectamine reagent (Life Technologies, Rockville, MD).
Generation of preproapelin transgenic mice
Briefly, rat preproapelin-encoding cDNA was inserted in the PcAGGS vector at the XhoI site. The vector’s backbone was subsequently removed by restriction enzyme digestion with StuI and ScaI. The remaining construct (550 bp) was purified and injected into mouse egg donor (strain C57BL/6xSJL) with multicopy insertion. Six litters were born (88 mice), of which 64 mice remained after 3 wk. Genotype analysis revealed 10 PcAGGS-positive mice. These mice were crossed with wild-type mice, and their litters were genotyped for PcAGGS vector.
Membrane preparation
All cells and tissues were washed extensively with PBS. Whole-cell lysates of COS-7 cells were prepared by polytron disruption in ice-cold 5:2 lysis buffer (containing 5 mM Tris-HCl, 2 mM EDTA buffer containing 5 μg/ml leupeptin, 10 μg/ml benzamidine, and 5 μg/ml soybean trypsin inhibitor). For mouse tissues, adult mice (wild-type and preproapelin transgenic) were killed and brain and peripheral tissues were removed and frozen on crushed dry ice. These samples were homogenized in ice-cold 5:2 lysis buffer using a tight pestle tissue grinder and were then centrifuged and disrupted using a sonicator. Protein levels were determined by the Bradford assay according to the manufacturer’s instructions (Bio-Rad, Hercules, CA).
Immunoblot analyses
Whole-cell and tissue lysates were treated with deoxyribonuclease and ribonuclease (0.5 μg/μl each; Marligen Biosciences, Inc., Ijamsville, MD) for 20 min at 37 C. Samples subjected to reducing conditions were treated with 50 mM dithiothreitol (DTT) and 100 mM iodoacetamide for 30 min at 60 C. Twenty-five micrograms of protein of lysates were solubilized in sample buffer consisting of 50 mM Tris-HCl (pH 6.5), 1% sodium dodecyl sulfate, 10% glycerol, 0.003% bromophenol blue, and 10% 2-mercaptoethanol. The samples were subjected to polyacrylamide gel electrophoresis with 10–20% acrylamide gels and electroblotted onto nitrocellulose as previously described (24). Apelin-36 immunoreactivity was revealed with a rabbit polyclonal antibody (catalog number G-057-15; Phoenix Pharmaceuticals, Inc., Belmont, CA) at a concentration of 500 ng/ml.
Blood pressure and heart rate bioassay
Animal experiments and care were approved by the University of Toronto Animal Care Committee. Male Wistar rats and SHR weighing 280–330 g and at approximately 15 wk of age (Charles River Laboratories, Wilmington, MA) were anesthetized with Inactin (Promonta, Hamburg, Germany) administered ip at 100 mg/kg body weight (B.W.). The Inactin solution was prepared fresh every experimental day at a concentration of 50 mg/ml saline (0.9% NaCl) and kept on ice. Rats were placed on a heating pad to maintain normal body temperature throughout the experimental procedure. Right carotid artery and left femoral vein were isolated by blunt dissection and cannulated with PE-50 polyethylene tubing and tied firmly in place. Blood pressure and heart rate were recorded via cannula inserted in the right carotid artery. The basal blood pressure for SHR was stabilized at 220/170 mm Hg systolic/diastolic blood pressure. Apelin peptides were injected in via PE-50 cannula fitted with a 23-gauge needle placed in the left femoral vein. Blood pressure was recorded using a pressure transducer connected to a physiograph (MK-III-S; Narco Bio-Systems, Austin, TX). The pressure recording system was calibrated each experimental day.
Injection procedure.
Each rat received iv injection of angiotensin II (30 ng/kg B.W.; A-9225; Sigma, St. Louis, MO) to determine responsiveness. Only those responding with an increase of 15–20 mm Hg systolic were used for the bioassay of apelin peptides (4). Rats received an equivolume of saline as control, and blood pressure and heart rate was monitored for 10 min before injection of apelin peptides. The apelin analogs tested included: apelin-13 (QRPRLSHKGPMPF), apelin-12 (RPRLSHKGPMPF), apelin-13(F13A) (QRPRLSHKGPMPA), apelin-13(13[D-Phe]) (QRPRLSHKGPMP[D-Phe]), and apelin-13(F13[D-Ala]) (QRPRLSHKGPMP[D-Ala]) (Advanced Protein Technology Centre, University of Toronto). The peptides were dissolved as a stock solution of 100 μg/ml in 0.9% saline and administered at 6–25 μg/kg B.W. The blood pressure responses to iv injections of apelin peptides were expressed as mean ± SE and reported as systolic/diastolic pressure, mean arterial pressure, and changes in heart rate. Statistical analyses between treatments were calculated by unpaired two-tailed Student’s t tests using GraphPad (San Diego, CA) software, with a value of P < 0.05 considered statistically significant.
Results
Detection of preproapelin
An apelin-36 antibody was used to detect apelin-immunoreactivity in tissues from wild-type mice, preproapelin transgenic mice, and in COS-7 cells expressing preproapelin. Apelin immunoreactivity was detected as a band of approximately 16 kDa in lysates of heart tissue obtained from wild-type mice (Fig. 1A, lane 2). This band was also observed in lysates of several brain regions, lung, kidney, liver, and spleen. Apelin immunoreactivity was detected as two bands at approximately 8 and 16 kDa in the transgenic mice (Fig. 1A, lane 3) and COS-7 cells (Fig. 1A, lane 1), which correlated with presumed monomeric and dimeric forms of preproapelin. Lysates from COS-7 cells were reduced using DTT treatment, which revealed only the 8-kDa band (Fig. 1B, lane 2). The absence of the 16-kDa preproapelin species after reduction by DTT indicated the preproapelin dimer is stabilized by disulfide bridges linking cysteine residues. Because only the dimeric form was observed in normal mice, these findings suggest preproapelin exists physiologically as a dimer. However, the absence of cysteine residues in mature apelin-36 and apelin-13 suggest these processed peptides are monomeric.
FIG. 1. Apelin-36 antibody immunoblot analyses. Immunoblot analysis of whole-cell lysates using the apelin-36 antibody. A, Shown are samples from COS-7 cells expressing rat preproapelin (lane 1) and heart tissue samples for a control mouse (lane 2) and a preproapelin-transgenic littermate (lane 3). B, Samples from COS-7 cells expressing rat preproapelin either untreated (lane 1) or treated with 50 mM DTT and 100 mM iodoacetamide for 30 min at 60 C (lane 2). For all lanes, 25 μg of protein was used. The immunoblots shown are representative of two independent experiments.
Blood pressure modulation by apelin analogs in rats
We were the first to report a hypotensive action of apelin-13 in normotensive rats (4). In this study, we examined the effects of apelin and apelin analogs in a hypertensive rat model. Recognition of the critical nature of the carboxyl-terminal phenylalanine for apelin activity in vitro (22, 23) and as the site of ACE2 cleavage (21) prompted our investigation into the role of this residue on apelin function in vivo. Because apelin was previously shown to have a very short-lived effect in lowering blood pressure, we hypothesized that substituting a D-isomer amino acid in the carboxyl-terminal position would inhibit ACE2-mediated cleavage of the apelin peptide (Table 1). We also determined whether an alanine substitution in the carboxyl terminus of apelin-13 would yield an apelin-specific antagonist, as was previously shown for angiotensin II and saralasin (Table 1B), an early angiotensin II antagonist (25).
TABLE 1. Sequence of apelin analogs and related peptides
We compared the hypotensive effects of apelin in SHR and Wistar rats. The basal resting blood pressure values for SHR and Wistar rats were approximately 220/170 and 140/110 mm Hg respectively. Overall, administration of both apelin-12 and apelin-13 resulted in immediate decreases of both systolic and diastolic blood pressure with maximal responses within 1 min of injection. Injections of apelin-12 iv in doses of 6, 9, and 15 μg/kg B.W. revealed that only the 15-μg/kg dose induced a decrease in mean arterial blood pressure (MABP). Apelin-13 (15 μg/kg) administered iv to SHR and Wistar rats resulted in approximately 60 and 30% reduction in MABP, respectively (Fig 2A). In contrast to this, 15 μg/kg apelin-12 revealed approximately 15 and 30% decreases in MABP in SHR and Wistar rats, respectively. Because the magnitude of effect was greatest for apelin-13 in the SHR (from a basal blood pressure of 217/167 to 128/93 mm Hg within 1 min after injection), we used this rat model to test various analogs of the apelin-13 peptide. Injection of 15 μg/kg of apelin-13(13[D-Phe]) induced a rapid decrease of MABP, similar to apelin-13. However, the duration of effect was similar to apelin-13, suggesting that the D-isomer conformation of the phenylalanine did not affect efficacy or time course of action. Administration of 15 μg/kg of apelin-13(F13A) and apelin-13(F13[D-Ala]) revealed no significant reduction of MABP from baseline (Fig. 2B), indicating a loss of apelin effect. Overall, apelin-13 and apelin-13(13[D-Phe]) were the most potent hypotensive agents in SHR, whereas apelin-13(F13A) and apelin-13(F13[D-Ala]) produced no significant changes in blood pressure (summarized in Fig. 2C). For apelin and all apelin analogs, iv administration did not induce a significant change in heart rate, as illustrated by a representative trace for apelin-13 (Fig. 2A).
FIG. 2. Effects of apelin peptides on MABP in anesthetized rats. A, Time-course changes in MABP (% basal) after iv administration of 15 μg/kg apelin-12 or apelin-13 into Wistar or SHR at time 0 (arrow). Included is a representative time-course in heart rate (% basal) after iv administration of 15 μg/kg apelin-13 into SHR (open circles), as plotted vs. the right y-axis (Heart Rate). Data are mean ± SEM. B, Time-course changes in MABP (% basal) after iv administration of 15 μg/kg apelin-13 analogs into SHR at time 0 (arrow). Data are mean ± SEM. C, Comparison of the maximum decrease in MABP (% basal) of the apelin analogs in SHR. *, P < 0.0001 vs. apelin-13(F13A) treatment.
Whereas apelin-13(F13A) did not alter blood pressure on its own, we speculated that a concomitant dose may antagonize the hypotensive effects of apelin-13, as shown for saralasin and angiotensin II. SHR responsive to 15 μg/kg of apelin-13 were allowed to recover for 40 min, after which a combined dose of 15 μg/kg apelin-13 and 30 μg/kg apelin-13(F13A) showed a complete blockade of apelin hypotensive action (Fig. 3A). To ensure this was not the result of desensitization to apelin-13, 15 μg/kg of apelin-13 was injected after another 40-min recovery period, which resulted in a similar decrease in blood pressure as the first apelin-13 injection. This antagonism by apelin-13(F13A) was dose dependent, as shown by the effects of increasing doses of 0.1, 3, 10, and 30 μg/kg of apelin-13(F13A) administered concomitantly with 15 μg/kg apelin-13 (Fig. 3B). Furthermore, antagonism by apelin-13(F13A) was observed as specific against apelin-13, because no significant difference was observed between the increases in MABP of SHR treated with 40 ng/kg angiotensin II with or without coinjection of 30 μg/kg apelin-13(F13A) (Fig. 3C).
FIG. 3. Apelin-13(F13A) is an antagonist for apelin-13 in vivo. A, Time-course changes in systolic (open squares) and diastolic (open triangles) blood pressure (mm Hg) after iv administration of 15 μg/kg apelin-13 (closed arrow) and 30 μg/kg apelin-13(F13A) (open arrow) into SHR. Data are mean ± SEM (n = 3). B, Dose response of apelin-13(F13A) iv administration at 0.1 (n = 3), 3 (n = 4), 10 (n = 5), and 30 (n = 5) μg/kg with a concomitant injection of apelin-13 (15 μg/kg) into SHR. Shown is the MABP (% basal) at maximum decrease within 1 min after injection. Data are mean ± SEM. C, Comparison of the increase in MABP (% over basal) after iv administration of angiotensin II (40 ng/kg) alone or angiotensin II (40 ng/kg) and apelin-13(F13A) (30 μg/kg) together. Data are mean ± SEM (n = 5) with no significant difference observed between the two treatments.
Discussion
In this report, we demonstrate that apelin can lower blood pressure in normotensive rats and to a much greater extent in the hypertensive SHR. The similar decreases in both systolic and diastolic blood pressure were indicative of an arterial vasodilation effect. Decrements in blood pressure were dramatically attenuated by coadministration of the apelin-13 analog with a single amino acid substitution of the carboxyl-terminal phenylalanine, yielding the successful development of a novel apelin antagonist. In addition, we observed preproapelin to be exclusively dimeric in vivo.
To compare the hypotensive effects of apelin in normotensive and hypertensive rats, we tested the effects of iv injections of apelin in Wistar rats and SHR. We observed no differences in the hypotensive effects between 15-μg/kg rat B.W. doses of apelin-12 and apelin-13 in Wistar rats. Another group reported apelin-12 as more potent than apelin-13 in reducing MABP in Wistar rats (15). These disparate results may perhaps be explained by the different anesthetic treatments used on these rats. Apelin-13 had a much greater potency than apelin-12 in SHR, decreasing MABP by approximately 60% compared with approximately 15% (i.e. respective reductions of 90 and 15 mm Hg). Due to the high sensitivity observed in SHR with apelin-13, we performed subsequent experiments using SHR to test the apelin-13 analogs.
As a basis for designing potential apelin antagonists, we examined an earlier successful strategy used for the closely related angiotensin II peptide. Before the development of angiotensin receptor subtype-specific antagonists and ACE inhibitors, an angiotensin II analog, saralasin (angiotensin II[Sar1, Val5, Ala8]) was found to act as an antagonist to angiotensin II pressor effects in rats (25). We hypothesized that a similar substitution of alanine in place of the carboxyl-terminal phenylalanine in apelin-13 would produce an effective antagonist. The dramatic reduction of blood pressure by apelin-13 was completely attenuated in a second injection combined with apelin-13(F13A). This effect was not due to apelin receptor desensitization, since a subsequent administration of apelin-13 displayed an identical reduction in blood pressure, indicating intact receptor sensitivity. Apelin-13(F13A) antagonism of apelin-13 was observed to be dose-dependent and did not affect the activity of angiotensin II on blood pressure, which suggests specific antagonism at the apelin receptor. Furthermore, the parallel changes in both systolic and diastolic blood pressure were evidence of both arterial and venous dilator effects, which corresponded with a previous study which described apelin-induced decreases in both MABP and mean circulatory filling pressure (16). The apelin-13(F13A) analog has been examined with varying results in vitro, with regard to ligand binding affinities and effect on second messenger systems (22, 23, 26). These in vitro differences have not yet been reconciled, whereas we observed a clear antagonist action in vivo.
Recently, a second angiotensin-converting enzyme-related carboxypeptidase (ACE2) was discovered to hydrolyze both angiotensin II and apelin, which resulted in cleavage of the carboxyl-terminal phenylalanine residues from both peptides. We hypothesized the inclusion a D-isomer carboxyl amino acid residue would prevent hydrolysis of apelin-13 and perhaps heighten or extend the hypotensive effects in vivo. However, little difference was observed between apelin-13 and apelin-13(13[D-Phe]) peptides, suggesting that the D-conformation of the carboxyl-terminal phenylalanine was irrelevant to the hydrolysis activity of ACE2.
In wild-type mice, preproapelin was detected as a dimeric protein. Monomeric preproapelin was only detected in cells and transgenic mice overexpressing preproapelin, suggesting that preproapelin exists exclusively in dimeric form in vivo. Reduction of dimeric to monomeric preproapelin indicated the dimerization was a consequence of disulfide bridges formed between cysteine residues. Sequence analysis revealed two cysteines flanking the signal sequence at the amino-terminal end of human and bovine preproapelin. These cysteines are conserved in rat preproapelin with an additional cysteine embedded within the signal sequence. However, no cysteines were observed in the processed apelin-36 and apelin-13 peptides, which are likely monomeric. Dimerization has been revealed as a prerequisite step for proper processing of other prepropeptides including prosomatostatin-II (27), prouroguanylin (28), and the human nerve growth factor (29).
In conclusion, we report preproapelin is dimeric in native tissues and in transfected cells. Functionally we demonstrate a pronounced hypotensive effect of apelin-13 in a hypertensive rate model compared with normotensive rats. We have developed a novel apelin receptor antagonist generated by an alanine substitution in the carboxyl-terminal end of apelin-13. Because there are no known selective antagonists for the apelinergic system, the identification of a highly selective apelin antagonist should greatly aid in the characterization of this receptor system and possibly lead to novel advances in apelin-related therapeutic treatment.
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