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Apelin

Apelin is an endogenous ligand of the human orphan receptor APJ (orphan G protein-coupled receptor). This peptide is produced through processing from the C-terminal portion in the pre-proprotein consisting of 77 amino acid residues and exists in multiple molecular forms. Although the main physiological functions of apelin have not been clarified yet, it has been demonstrated that apelin partially suppresses cytokine production from mouse spleen and, specifically, induces the promotion of extracellular acidification and inhibition of cAMP production in Chinese hamster ovary cells. Moreover, it is involved in the regulation of blood pressure and blood flow.

De Falco et al. In Vivo 2002 Sep-Oct;16(5):333-6

Apelin gene encodes a pre-proprotein of 77 amino acids, with a signal peptide in the N-terminal region. After translocation into the endoplasmic reticulum and cleavage of the signal peptide, the proprotein of 55 amino acids may generate several active fragments: a 36 amino acid peptide corresponding to the sequence 42-77 (apelin 36), a 31 amino acid peptide corresponding to the sequence 47-77 (apelin 31), a 28 amino acid peptide corresponding to the sequence 50-77 (apelin 28) and a 13 amino acid peptide corresponding to the sequence 65-77 (apelin 13). This latter fragment may also undergo a pyroglutamylation at the level of its N-terminal glutamine residue. However the presence and/or the concentrations of those peptides in human plasma has been questioned. Recently, 46 different apelin peptides ranging from apelin 55 (proapelin) to apelin 12 have been identified in bovine colostrum, including C-ter truncated isoforms.

 

1 1 1
 

Binding Characteristics of [125I]-[Pyr1]-Apelin-13 in Human Heart Tissue

Kd (nM) Bmax (fmol/mg Protein)
Left Ventricle 0.35±0.08  4.3±0.9 
Right Atria 0.33±0.09 3.1±0.6

Distribution of APJ Orphan Receptor in Human Cardiovascular Tissue

Tissue

Binding Density of [125I]-[Pyr1]-Apelin-13 (autography)

Heart (LV & RA)

++++

Coronary artery

+++

Aorta

+++

Saphenous Vein Grafts

+++

Distribution of APJ Orphan Receptor in Rat Tissues

Tissue

Binding Density of [125I]-[Pyr1]-Apelin-13 (autography)

Cerebellum

+++

Lung

+++

Heart

+++

Kidney Cortex

+

Katugampola et al. Br J Pharmacol. 2001 Mar;132(6):1255-60.
 
1 1 1

Apelin, the novel endogenous ligand of the orphan receptor APJ, regulates cardiac contractility

The orphan receptor APJ and its recently identified endogenous ligand, apelin, exhibit high levels of mRNA expression in the heart. However, the functional importance of apelin in the cardiovascular system is not known. In isolated perfused rat hearts, infusion of apelin (0.01 to 10 nmol/L) induced a dose-dependent positive inotropic effect (EC50: 33.1+/-1.5 pmol/L). Moreover, preload-induced increase in dP/dt(max) was significantly augmented (P<0.05) in the presence of apelin. Inhibition of phospholipase C (PLC) with U-73122 and suppression of protein kinase C (PKC) with stauzrosporine and GF-109203X markedly attenuated the apelin-induced inotropic effect (P<0.001). In addition, zoniporide, a selective inhibitor of Na+-H+ exchange (NHE) isoform-1, and KB-R7943, a potent inhibitor of the reverse mode Na+-Ca2+ exchange (NCX), significantly suppressed the response to apelin (P<0.001). Perforated patch-clamp recordings showed that apelin did not modulate L-type Ca2+ current or voltage-activated K+ currents in isolated adult rat ventricular myocytes. Apelin mRNA was markedly downregulated in cultured neonatal rat ventricular myocytes subjected to mechanical stretch and in vivo in two models of chronic ventricular pressure overload. The present study provides the first evidence for the physiological significance of apelin in the heart. Our results show that apelin is one of the most potent endogenous positive inotropic substances yet identified and that the inotropic response to apelin may involve activation of PLC, PKC, and sarcolemmal NHE and NCX.

Szokodi et al.Circ Res 2002 Sep 6;91(5):434-40

 

The novel peptide apelin lowers blood pressure via a nitric oxide-dependent mechanism

Apelin is an endogenous ligand of the human orphan receptor APJ. We detected apelin-like immunoreactivity in the adipocytes, gastric mucosa, and Kupffer cells in the liver. We also detected apelin-like immunoreactivity localized within the endothelia of small arteries in various organs. Further, it was found that mean arterial pressure after the administration of apelin-12, apelin-13, and apelin-36 at a dose of 10 nmol/kg in anaesthetized rats was reduced by 26+/-5, 11+/-4, and 5+/-4 mm Hg, respectively. In the presence of a nitric oxide (NO) synthase inhibitor, the effect of apelin-12 on blood pressure was abolished. Furthermore, the administration of apelin-12 (10 nmol/kg) in rats produced a transitory elevation of the plasma nitrite/nitrate concentration from a basal level of 21.4+/-1.6 to 27.0+/-1.5 microM. Thus, apelin may lower blood pressure via a nitric oxide-dependent mechanism.

Tatemoto et al. Regul Pept 2001 Jun 15;99(2-3):87-92

 

Apelin-immunoreactivity in the rat hypothalamus and pituitary

With the use of an antiserum against human apelin-36 (Phoenix Pharmaceuticals), apelin-immunoreactivity (irAP) was detected in neurons and cell processes of the supraoptic nucleus (SO), paraventricular nucleus (PVH), accessory neurosecretory nuclei (Acc) and suprachiasmatic nucleus. Strongly labeled cells/processes were noted in the internal layer of the median eminence, infundibular stem, anterior and posterior pituitary. Double-labeling the sections with goat polyclonal neurophysin I-antiserum and rabbit polyclonal apelin-antiserum revealed a population of magnocellular neurons in the PVH, SO and Acc expressing both irAP and neurophysin I-immunoreactivity (irNP), the latter being a marker of oxytocin-containing neurons. By inference, the AP-positive but irNP-negative magnocellular neurons could be vasopressin-containing. The presence of irAP in certain hypothalamic nuclei and pituitary suggests that the peptide may be a signaling molecule released from the hypothalamic-hypophysial axis.

Brailoiu et al. Neurosci Lett 2002 Jul 26;327(3):193-7

 

The effects of centrally administered apelin-13 on food intake, water intake and pituitary hormone release in rats

Apelin is the recently identified endogenous ligand for the G-protein-coupled receptor, APJ. Preproapelin and APJ mRNA are found in hypothalamic regions known to be important in the regulation of food and water intake, and pituitary hormone release. The effects of intracerebroventricular (ICV) administration of pyroglutamylated apelin-13 on food and water intake and pituitary hormone release in rats were investigated. Apelin-13 had little effect on food intake, but dose-dependently increased drinking behaviour and water intake at 1 h. Apelin-13 (10 nmol) increased water intake by up to sixfold compared to saline. Compared to saline control, apelin-13 (10 nmol) significantly increased plasma ACTH and corticosterone and decreased plasma prolactin, LH and FSH at 30 min. In vitro, apelin-13 stimulated the release of CRH and AVP from hypothalamic explants, but had no effect on NPY release. These results suggest that apelin may play an important role in the hypothalamic regulation of water intake and endocrine axes.

Taheri et al. Biochem Biophys Res Commun 2002 Mar 15;291(5):1208-12

 

Apelin, orexin-A and leptin plasma levels in morbid obesity and effect of gastric banding

Maintenance of human energy homeostasis is regulated by a complex network. Peptides secreted from the gastrointestinal tract (GI) are signaling to the brain and other organs initiating or terminating food intake and energy expenditure. In the present study we investigated basal plasma levels of apelin, orexin-A, and leptin in morbid obese patients. In addition, we measured in a subgroup of these patients in the same individual orexin-A and leptin plasma levels one year after gastric banding surgery. METHODS: Basal plasma values were determined in obese patients (BMI=48+/-1 kg/m2n=32) after an overnight fast and compared to healthy, normal weighted (BMI=22+/-2 kg/m2n=12) controls. In addition, blood samples were collected in a subgroup of patients (BMI=48+/-1 kg/m2n=8) the day before surgery and 1 year after the operation. Apelin, orexin-A, and leptin levels were analysed using ELISAs. RESULTS: One year after the operation obese patients significantly lost weight (from 48+/-2 kg/m2 to 39+/-2 kg/m2; p<0,001). Apelin, orexin-A and leptin levels in obese patients were significantly higher compared to control individuals (736+/-50 pg/ml vs. 174+/-14 pg/ml, p<0.0001; 75.3+/-24.1 pg/ml vs. 0.8+/-0.4 pg/ml, p<0.0001; 79.0+/-2.4 ng/ml vs. 5.8+/-0.8 ng/ml, p<0.0001, respectively). Apelin and leptin plasma concentrations also correlated significantly with BMI (r=0.769, p<0.0001; r=0.778; p<0.0001, respectively), while orexin-A correlation was rather weak (r=0.335, p<0.03). No difference between pre- and post-operative orexin-A levels was observed, while leptin plasma levels significantly decreased from 45.1+/-5.4 ng/ml to 27.3+/-6.0 ng/ml (p=0.015). CONCLUSIONS: Apelin, orexin-A, and leptin plasma levels correlated positively with the BMI. One year after gastric banding with significant loss in BMI basal plasma levels of leptin decreased, while orexin-A remained unchanged.

Basal apelin plasma concentration in obese patients

apelin-36

Plasma apelin levels correlated significantly with BMI (r = 0.769; p = 0.0001). Mean value in obese patients (n = 25; 736 ± 50 pg/ml) was significantly higher compared to controls (n = 12; 174 ± 14 pg/ml, p < 0.0001).

Heinonen MV, et al. Regul Pept. 2005 Aug 15;130(1-2):7-13.

 

Apelin, a new enteric peptide: localization in the gastrointestinal tract, ontogeny, and stimulation of gastric cell proliferation and of cholecystokinin secretion.

Apelin is a recently discovered peptide that is the endogenous ligand for the APJ receptor. The aim of this study was to characterize apelin expression (mRNA levels) in the rat gastrointestinal (GI) tract and pancreas, to localize distribution of apelin peptide containing cells in the stomach by immunohistochemistry (IHC), to characterize the ontogeny of gastric apelin expression and peptide, and the influence of apelin on gastric cell proliferation in vitro. Additionally, the effect of apelin on cholecystokinin (CCK) secretion, and the involvement of mitogen-activated protein kinase (MAPK), protein kinase C (PKC) and changes in [Ca++]i in apelin-induced CCK secretion in vitro were examined. Northern analysis showed a maximal apelin expression in the stomach with a lower expression level in the intestine. Apelin expression was not detected in the pancreas. IHC revealed abundant apelin positive cells in the glandular epithelium of the stomach. The ontogeny study showed a higher apelin expression in the fetal and postnatal rat stomachs when compared to the adult stomach. In contrast to apelin expression, apelin peptide was not detected in the rat stomach until 20 days of age and then increased progressively with age. Apelin was shown to stimulate gastric cell proliferation in vitro. Apelin also stimulated CCK secretion from a murine enteroendocrine cell line (STC-1); apelin-stimulated CCK secretion is mediated through MAPK but not by [Ca++]i signaling. Together, these data indicate that apelin is an important new stomach peptide with a potential physiologic role in the GI tract.

Wang et al. Endocrinology. 2004 Mar;145(3):1342-8.

 

Apelin, a newly identified adipokine up regulated by insulin and obesity

The results presented herein demonstrate that apelin is expressed and secreted by both human and mouse adipocytes. Apelin mRNA levels in isolated adipocytes are close to other cell types present in white adipose tissue or other organs known to express apelin such as kidney, heart, and to a lesser extent brown adipose tissue. Apelin expression is increased during adipocyte differentiation stage. A comparison of four different models of obesity in mice showed a large increase in both apelin expression in fat cells and apelin plasma levels in all the hyperinsulinemia-associated obesities and clearly demonstrated that obesity or high fat feeding are not the main determinants of the rise of apelin expression. The lack of insulin in streptozotocin-treated mice is associated with a decreased expression of apelin in adipocytes. Furthermore, apelin expression in fat cells is strongly inhibited by fasting and recovered after refeeding, in a similar way to insulin. A direct regulation of apelin expression by insulin is observed in both human and mouse adipocytes and clearly associated with the stimulation of PI3K, PKC and MAPK. These data provide evidence that insulin exerts a direct control on apelin gene expression in adipocytes. In obese patients, both plasma apelin and insulin levels were significantly higher suggesting that the regulation of apelin by insulin could influence blood concentrations of apelin. The present work identifies apelin as a novel adipocyte endocrine secretion and focuses on its potential link with obesity-associated variations.

Boucher et al. Endocrinology. 2005 Apr;146(4):1764-71.

 

Modification of the terminal residue of apelin-13 antagonizes its hypotensive action.

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 microg/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 microg/kg) blocked hypotensive effects of apelin-13 (15 microg/kg), which revealed that apelin-13(F13A) behaved as an apelin-specific antagonist.

Lee et al. Endocrinology. 2005 Jan;146(1):231-6.

 

Circulating and cardiac levels of apelin, the novel ligand of the orphan receptor APJ, in patients with heart failure

The orphan receptor APJ and its recently identified endogenous ligand, apelin, are expressed in the heart. However, their importance in the human cardiovascular system is not known. This study shows that apelin-like immunoreactivity is abundantly present in healthy human heart and plasma. Gel filtration HPLC analysis revealed that atrial and plasma levels of high molecular weight apelin, possibly proapelin, were markedly higher than those of mature apelin-36 itself. As assessed by quantitative RT-PCR analysis, left ventricular apelin mRNA levels were increased 4.7-fold in chronic heart failure (CHF) due to coronary heart disease (p<0.01) and 3.3-fold due to idiopathic dilated cardiomyopathy (p<0.05), whereas atrial apelin mRNA levels were unchanged. Atrial and plasma apelin-like immunoreactivity (using Phoenix's Apelin-36 (Human) RIA Kit) as well as atrial and ventricular APJ receptor mRNA levels were significantly decreased in CHF. Our results suggest that a new cardiac regulatory peptide, apelin, and APJ receptor may contribute to the pathophysiology of human CHF.

Atrial and ventricular immunoreactive apelin levels

Apelin-like immunoreactivity was detected in the heart of organ donors, being over 200-fold higher in the right atria than in the left ventricles (650 ± 145 pg/mg, n=5 and 2.8 ± 0.6 pg/mg, n=10, respectively). There was a tendency for left ventricular ir-apelin levels to be higher in patients with heart failure due to coronary heart disease (4.4 ± 0.5 pg/mg, p=0.07) and idiopathic dilated cardiomyopathy (3.8 ± 0.9 pg/mg, p=0.4) than those in controls, but these changes were not statistically significant (Fig. 1B). Interestingly, atrial ir-apelin levels were significantly decreased in patients with heart failure (Fig. 2B).

Plasma apelin levels in normal subjects and in patients with heart failure

Immunoreactive apelin was found to be present in normal human plasma (mean, 89.8 ± 5.3 pg/ml, n=6). Plasma levels of ir-apelin were significantly decreased in patients with heart failure due to coronary heart disease compared to normal subjects (in NYHA III: 71 ± 6 pg/ml, p<0.05). Plasma ir-apelin levels showed significant correlation to atrial ir-apelin levels (R=0.4, n=38, p<0.05).

Fig. 01

Fig. 02

Left ventricular apelin mRNA (A), immunoreactive apelin-36 (B), and APJ receptor mRNA (C) levels in control subjects (n=9) and patients with end-stage heart failure due to coronary heart disease (CHD, n=7) and idiopathic dilated cardiomyopathy (IDC, n=6). The mRNA results are expressed as ratios to 18S RNA determined by TaqMan real-time quantitative RT-PCR analysis. Bars indicate means ?nbsp;SEM. *p<0.05 and **p<0.01 vs. control subjects.

Right atrial apelin mRNA (A), immunoreactive apelin-36 (B), and APJ receptor mRNA (C) levels in control subjects (n=6) and patients with heart failure (NYHA functional class II or III) due to coronary heart disease (CHD, n=38). Bars indicate means ?nbsp;SEM. *p<0.05 and **p<0.01 vs. control subjects.

Fig. 03

Gel filtration HPLC analysis of immunoreactive apelin in plasma (A,B) and atrial extracts (C,D) of a healthy control (A,C) and a patient with heart failure (B,D). The arrows denote elution positions of bovine serum albumin (V0), apelin-36, and 125I-, used for the calibration of the column.

IDC, idiopathic dilated cardiomyopathy; CCHD, coronary heart disease.

Foldes G, et al. Biochem Biophys Res Commun. 2003 Aug 29;308(3):480-5

 

Apelin expression in normal human tissues

Apelin is an endogenous ligand of the human orphan receptor APJ (orphan G protein-coupled receptor). This peptide is produced through processing from the C-terminal portion in the pre-proprotein consisting of 77 amino acid residues and exists in multiple molecular forms. Although the main physiological functions of apelin have not been clarified yet, it has been demonstrated that apelin partially suppresses cytokine production from mouse spleen and, specifically, induces the promotion of extracellular acidification and inhibition of cAMP production in Chinese hamster ovary cells. Moreover, it is involved in the regulation of blood pressure and blood flow. In this study we have analyzed, by immunohistochemistry, apelin distribution in several human tissues, demonstrating that apelin has a widespread pattern of expression. These results seem to confirm that apelin functions widely in various tissues interacting with its specific receptor APJ.

Tissue and blood sampling. After removal, cardiac tissue samples were blotted dry, immersed in liquid nitrogen, and stored at -80 C until assayed. For plasma sampling, blood was taken before surgery after 30 min bed rest and collected into chilled tubes containing EDTA. The plasma was separated by centrifugation at +4 oC and kept at -80 oC until assayed.

Radioimmunoassay for apelin. Immunoreactive apelin (ir-apelin) was determined from extracted plasma and right atrial and left ventricular samples using an apelin-36 radioimmunoassay kit (Phoenix, Cat-No. RK-057-15). Apelin assay was performed according to the manufacturer's instructions. The sensitivity of assay was 1 fmol/tube. Tissue peptide levels are expressed as a concentration per mg wet weight.

HPLC analysis. Gel filtration high performance liquid chromatography (GF-HPLC) and apelin radioimmunoassay were used in order to characterize the molecular form of immunoreactive apelin in plasma and tissue extracts. Aliquots of the atrial tissue homogenates, used for RNA extraction, from healthy subjects and patients with heart failure were diluted to 400 ul of 40% acetonitrile in aqueous 0.1% TFA. Plasma samples (1 ml) were extracted with SepPak C18 cartridges, dried, and reconstituted in 400 ul of 40% acetonitrile in aqueous 0.1% TFA. The samples were passed through Millex HV filters (Millipore) before being loaded into the 7.8 ?nbsp;300 mm ProteinPak-125 column (Waters). The column was eluted with 40% acetonitrile in aqueous 0.1% TFA at 1 ml/min. Fractions of 0.5 ml were collected, dried in Savant SpeedVac, and subjected in duplicate to apelin radioimmunoassay. The column was calibrated with bovine serum albumin (void volume), apelin-36, and 125I- (total volume).

De Falco M, et al. In Vivo 2002 Sep-Oct;16(5):333-6

 

Characterization of apelin, the ligand for the APJ receptor.

The apelin peptide was recently discovered and demonstrated to be the endogenous ligand for the G protein-coupled receptor, APJ. A search of the GenBank databases retrieved a rat expressed sequence tag partially encoding the preproapelin sequence. The GenBank search also revealed a human sequence on chromosome Xq25-26.1, containing the gene encoding preproapelin. We have used the rat sequence to screen a rat brain cDNA library to obtain a cDNA encoding the full-length open reading frame of rat preproapelin. This cDNA encoded a protein of 77 amino acids, sharing an identity of 82% with human preproapelin. Northern and in situ hybridization analyses revealed both human and rat apelin and APJ to be expressed in the brain and periphery. Both sequence and mRNA expression distribution analyses revealed similarities between apelin and angiotensin II, suggesting they that share related physiological roles. A synthetic apelin peptide was injected intravenously into male Wistar rats, resulting in immediate lowering of both systolic and diastolic blood pressure, which persisted for several minutes. Intraperitoneal apelin injections induced an increase in drinking behavior within the first 30 min after injection, with a return to baseline within 1 h.

Lee et al. J Neurochem. 2000 Jan;74(1):34-41.

Northern blot analysis of the distribution of preproapelin mRNA in human (A) and rat (B and C) tissues. Each lane contained 10 ug of poly(A)+ RNA isolated from various tissues, with the exception of rat pituitary (7.5 ug), olfactory tubercle (7.8 ug), septum (5 ug), and liver (7 ug).

Darkfield autoradiograms of coronal sections of rat brain showing the localization of preproapelin mRNA, at various distances from bregma according to the stereotactic coordinates of Paxinos and Watson (1982). Shown are representative sections at levels relative to the bregma at -0.3 mm (A), -0.5 mm (B), -1.8 mm (C), -2.8 mm (D), -3.9 to -4.2 mm (E), and -5.8 mm (F). 3v, third ventricle; 4v, fourth ventricle; 7, facial nucleus; AC, anterior cingulate cortex; AD, anterodorsal thalamic nucleus; AM, anteromedial thalamic nucleus; APT, anterior pretectal area; AUD, auditory area; CA, field of Ammon's horn; Ch, choroid plexus; CL, centrolateral thalamic nucleus; CP, caudate putamen; DG, dentate gyrus; DK, nucleus of Darkschewitsch; DM, dorsomedial hypothalamic nucleus; FP, frontoparietal cortex; IC, inferior colliculus; IPN, interpeduncular nucleus; LG, lateral geniculate complex; LS, lateral septal nucleus; MA, magnocellular preoptic nucleus; MD, mediodorsal thalamic nucleus; MEA, medial amygdaloid nucleus; MEPO, median preoptic nucleus; MG, medial geniculate nucleus; MH, medial habenular nucleus; MPO, medial preoptic area; OB, olfactory bulb; OT, olfactory tubercle; PAG, periaqueductal gray; PB, parabrachial nucleus; PE, periventricular hypothalamic nucleus; Pi, pineal gland; PO, primary olfactory cortex; PT, parietal region; PVN, paraventricular hypothalamic nucleus; RS, retrosplenial area; S5, sensory root of the trigeminal nerve; SC, superior colliculus; SF, septofimbrial nucleus; SH, septohypothalamic nucleus; SO, supraoptic hypothalamic nucleus; SS, primary somatosensory area; ST/STH, subthalamic nucleus; SUB, dorsal subiculum; TS, triangular septal nucleus; V, vestibular nucleus; VAL, ventral anterior-lateral complex thalamus; VIS, primary visual area; VM, ventromedial thalamic nucleus; VP, ventroposterior thalamic nucleus; ZI, zona incerta.

 

Darkfield autoradiograms of sagittal and coronal sections of rat brain showing the localization of preproapelin mRNA (A) and APJ receptor mRNA (B-E). A and B show lateral representative sections at 1.77 and 0.4 mm, respectively. Also shown are representative sections at levels relative to the bregma at 1.2 mm (C), -1.8 mm (D), and -4.8 mm (E). See Fig. 3 for abbreviation definitions.

 

Blood pressure and heart rate changes after 1 and 2 g/300 g B.W. intravenous apelin injection into rats (n = 5). Values are shown as means ?SEM.

 

Water consumption after intraperitoneal injection of 100 ug of apelin into rats (n = 18), recorded at 30-min intervals. An asterisk indicates a significant difference in water consumption between vehicle- and apelin-injected rats. Values are shown as means ?SEM.

Lee, Dennis K., et al. Characterization of Apelin, the Ligand for the APJ Receptor. Journal of Neurochemistry 74 (1), 34-41.

 

Search for an endogenous ligand of the orphan G protein-coupled receptor--discovery of apelin, a novel biologically active peptide.

In the search for an endogenous ligand of the human orphan receptor APJ, we have isolated from bovine stomach extracts a novel 36-amino acid peptide, designated apelin. The APJ receptor is one of the G protein-coupled orphan receptors, many of which have been considered to be specific receptors for unidentified hormones and neuropeptides. We recently found the presence of apelin in the adipocytes and vascular walls as well as in the stomach. We examined biological activities of apelin and found that apelin lowered blood pressure in rats and also released cholecystokinin(CCK) from dispersed intestinal endocrine cells. Since apelin is an endogenous ligand for the HIV entry coreceptor APJ, we tested the effect of apelin on the entry of HIV in association with CD4, and found that apelin blocked the entry of HIV-1 and HIV-2.

Takemoto et al. Nippon Rinsho. 2000 Mar;58(3):737-46.

 

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 apelin-36 was sustained. These responses were almost completely inhibited by a specific inhibitor for G(i) or that for Na(+)/H(+) 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 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 [<Glu(65)]apelin-13 was more potent than apelin-36 in this activity. Heterogeneous molecular forms of apelin corresponding to apelin-36 and [<Glu(65)]apelin-13 were produced in bovine colostrum. Apelin-36 and [<Glu(65)]apelin-13 might have different functions in vivo and in vitro.

Hosoya et al. J Biol Chem. 2000 Jul 14;275(28):21061-7.

 

Apelin peptides block the entry of human immunodeficiency virus (HIV).

The orphan G protein-coupled receptor APJ has been shown to be a coreceptor for human and simian immunodeficiency virus (HIV and SIV) strains. We have determined that some HIV and SIV strains use APJ as a coreceptor to infect the brain-derived NP-2/CD4 cells. Because apelin is an endogenous ligand for the APJ receptor, we examined the inhibitory effects of apelin peptides on HIV infection, and found that the apelin peptides inhibit the entry of some HIV-1 and HIV-2 into the NP-2/CD4 cells expressing APJ. The inhibitory efficiency has been found to be in the order of apelin-36>apelin-17>apelin-13>apelin-12.

Zou et al. FEBS Lett 2000 May 4;473(1):15-8.

 

Characterization of apelin, the ligand for the APJ receptor.

The apelin peptide was recently discovered and demonstrated to be the endogenous ligand for the G protein-coupled receptor, APJ. A search of the GenBank databases retrieved a rat expressed sequence tag partially encoding the preproapelin sequence. The GenBank search also revealed a human sequence on chromosome Xq25-26.1, containing the gene encoding preproapelin. We have used the rat sequence to screen a rat brain cDNA library to obtain a cDNA encoding the full-length open reading frame of rat preproapelin. This cDNA encoded a protein of 77 amino acids, sharing an identity of 82% with human preproapelin. Northern and in situ hybridization analyses revealed both human and rat apelin and APJ to be expressed in the brain and periphery. Both sequence and mRNA expression distribution analyses revealed similarities between apelin and angiotensin II, suggesting they that share related physiological roles. A synthetic apelin peptide was injected intravenously into male Wistar rats, resulting in immediate lowering of both systolic and diastolic blood pressure, which persisted for several minutes. Intraperitoneal apelin injections induced an increase in drinking behavior within the first 30 min after injection, with a return to baseline within 1 h.

Lee et al. J Neurochem. 2000 Jan;74(1):34-41.

 

Apelin, the natural ligand of the orphan receptor APJ, is abundantly secreted in the colostrum.

By using a strategy that we have developed to search for the ligands of orphan seven-transmembrane-domain receptors [S. Hinuma et al., Nature 393 (1998) 272-276], we have recently identified a natural ligand, apelin, for the orphan 7TMR, APJ [K. Tatemoto et al., Biochem. Biophys. Res. Commun. 251 (1998) 471-476]. In this paper, we isolated rat and mouse apelin cDNAs, and analyzed the tissue distribution of apelin mRNA in rats. Although apelin mRNA was widely detected in a variety of tissues, the highest expression of apelin mRNA was detected in the mammary gland of pregnant rats. In the mammary gland, biologically active apelin and its mRNA considerably increased during pregnancy and lactation, and reached a maximal level around parturition. Moreover, a large amount of apelin (14-93 pmol/ml) was found to be secreted in the bovine colostrum, and it was still detectable even in commercial bovine milk. Since apelin partially suppressed cytokine production by mouse spleen cells in response to T cell receptor/CD3 cross-linking, the oral intake of apelin in the colostrum and milk might modulate immune responses in neonates.

Habata et al. Biochim Biophys Acta. 1999 Oct 13;1452(1):25-35.

 

[(125)I]-(Pyr(1))Apelin-13 is a novel radioligand for localizing the APJ orphan receptor in human and rat tissues with evidence for a vasoconstrictor role in man.

1. We have determined the binding characteristics of [(125)I]-(Pyr(1))Apelin-13, a putative ligand for the APJ orphan receptor in human cardiovascular and rat tissue and investigated the functional properties of (Pyr(1))Apelin-13 in human saphenous vein. 2. The binding of [(125)I]-(Pyr(1))Apelin-13 to sections of human heart tissue was time dependent and rapid at 23 degrees C. Data were fitted to a single site model with an association rate constant (k(obs)) of 0.115 min(-1). [(125)I]-(Pyr(1))Apelin-13 also dissociated from a single site with a dissociation rate constant of 0.0105 min(-1). 3. In saturation binding experiments [(125)I]-(Pyr(1))Apelin-13 bound to human left ventricle with a K(D) value of 0.35+/-0.08 nM, B(max) of 4.3+/-0.9 fmol mg(-1) protein with a Hill slope of 0.97+/-0.04 and to the right atria with a K(D) of 0.33+/-0.09 nM, B(max) of 3.1+/-0.6 fmol mg(-1) protein and a Hill slope of 0.93+/-0.05. 4. [(125)I]-(Pyr(1))Apelin-13 binding sites were localized using autoradiography to human cardiovascular tissue, including coronary artery, aorta and saphenous vein grafts. In rat tissue a high density of receptors were localized to the molecular layer of the rat cerebellum, rat lung, rat heart and low levels in the rat kidney cortex. 2. (Pyr(1))Apelin-13 potently contracted human saphenous vein with a pD(2) value of 8.4+/-0.2 (n=8). The maximum response elicited by the peptide was 22.6+/-6% of 100 mM KCl. 6. We provide the first evidence of APJ receptor expression, relative densities and functional properties of (Pyr(1))Apelin-13 in human cardiovascular tissue.

Katugampola et al. Br J Pharmacol. 2001 Mar;132(6):1255-60.

 Dr. Nae J. Dun, 

East Tennessee State University

Tissue Sample Rat Hypothalamus
Fixative 4% paraformaldehyde/ 0.2% picric acid in PBS
Embedding paraffin
Control No primary antibody and pre-absorption of the antibody with the peptide apelin-36 (1µg/ml)
Blocking 2% Normal Goat Serum
Primary Antibody Anti-Apelin-36 (Human) Antibody (Catalog No.: H-057-15)
Optimal Dilution 1:3000, overnight at 4ºC
Secondary Antibody Goat Anti-Rabbit IgG, Biotinylated (1:50), 30 min
Amplification Streptavidin-HRP (Vector), 1:400, 30 min
Detection System HRP
Substrate DAB (Sigma), 3 min

Rat hypothalamus was stained by
Anti-Apelin-36 (Human) Serum (H-057-15


Mapping in Human Placenta tissue with Apelin-36 (Human) (H-057-15) and (Rat) (H-057-28) Antibodies
apelin  
Adult rat stomach and human stomach were stained using Apelin-36 (Human) antiserum (H-057-15)

Left. IHC localization of apelin containing cells in the oxyntic mucosa of the adult rat stomach. Strong apelin staining (red-brown) is observed in the deep glandular epithelium of the fundus.

Middle. A high-power magnification of stomach apelin containing cells.

Right
. IHC localization of apelin containing cells in the adult human stomach. This tissue was devoid of any pathophysiology. Apelin staining (brown) is observed in the glandular epithelium. No staining was observed with use of pre-immune serum (not shown). Micrographs were captured with a Nikon Eclipse E600 microscope with Plan Fluor, X100, X200, X200, respectively. L= stomach lumen, P= region of gastric pits, M= muscle layer of stomach.

Wang et al. Endocrinology. 2004 Mar;145(3):1342-8.


Rat stomach gastric gland was stained using Apelin-36 (Human) antiserum (H-057-15)

High Power micrograph showing apelin storage vesicles in a gastric gland of rat stomach. Apelin-staining vesicles are packed adjacent to the lumen (L) of the gastric gland suggesting release of apelin into the stomach lumen, X400.

Wang et al. Endocrinology. 2004 Mar;145(3):1342-8.

 

Ontogeny of apelin peptide in the oxyntic mucosa of the developing rat stomach.

Ontogeny of apelin peptide in the oxyntic mucosa of the developing rat stomach. Apelin containing cells (brown color) were observed initially in the rat stomach at 20 days of age by IHC. IHC failed to detect apelin peptide in stomach specimens harvested from pups at 2, 7 and 13 days of age (data not shown). The number of apelin containing cells increased progressively with age.

Photos were captured with a 10X objective, M = muscle layer of stomach; E = gastric mucosal epithelium

Oxyntic mucosa of the developing rat stomach was stained using Phoenix's anti-Apelin-36 (Human) antiserum (Catalog Number: H-057-15).

Wang et al. Endocrinology. 2004 Mar;145(3):1342-8.

 

Ontogeny of apelin expression (mRNA levels) in the developing rat stomach

Top: Ontogeny of apelin expression (mRNA levels) in the developing rat stomach. Northern hybridization shows the profile of apelin expression in the fetal and postnatal rat stomachs compared with the adult rat stomach. Notice that apelin expression is barely discernible in the adult stomach on this film exposure.

Bottom: Real-time RT-PCR analysis of apelin expression in the fetal, postnatal, and adult rat stomach.

Wang et al. Endocrinology. 2004 Mar;145(3):1342-8.

 

Apelin-like immunoreactivity in vascular endothelial cells of large conduit vessels.
apelin

Apelin-like immunoreactivity (Apelin-12 (Human, Mouse, Rat, Bovine) Antiserum, Catalog No.: H-057-23, Phoenix Pharmaceuticals) vascular endothelial cells of large conduit vessels. Photomicrographs show staining in saphenous vein (a) and coronary artery (c) with higher magnification of the respective vessels in (b) and (d).

Kleinz et al. Regul Pept. 2004 May 15;118(3):119-25.

Western Blot analysis of Apelin in rat lung tissue by Apelin 12 (WBK-057-23) Antiserum
Mapping in Rat brain tissue with Apelin-12 (H-057-23) Serum

 

Apelin-12-ir in rat hypothalamus

Tissue Sample Rat lateral hypothalamus
Fixative 10% Formalin
Embedding Paraffin
Negative control No primary antibody
Pretreatment Target Retrieval 25 min (Steam)
Blocking 2% Normal Goat Serum
Primary Antibody Rabbit Anti-Apelin-12 (Human, Bovine) Antiserum (Catalog No.:H-057-23)
Optimal Dilution 1:3000 (1hour at RT)
Secondary Antibody Goat anti-Rabbit IgG, Biotinylated (1:400)
Amplification  Streptavidin-HRP (Vector), 1:400, 30 min
Detection system HRP
Substrate DAB (Sigma)
Counterstained Hematoxylin
Apelin-12-ir in rat  medial forebrain boundle
Tissue Sample Rat medial forebrain boundle
Fixative 10% Formalin
Embedding Paraffin
Negative control No primary antibody
Pretreatment Target Retrieval 25 min (Steam)
Blocking 2% Normal Goat Serum
Primary Antibody Rabbit Anti-Apelin-12 (Human, Bovine) Antiserum (Catalog No.:H-057-23)
Optimal Dilution 1:3000 (1hour at RT)
Secondary Antibody Goat anti-Rabbit IgG, Biotinylated (1:400)
Amplification  Streptavidin-HRP (Vector), 1:400, 30 min
Detection system HRP
Substrate DAB (Sigma)
Counterstained Hematoxylin
Photomicrographs demonstrating apelin-like immunoreactivity

a and d

e and f

Photomicrographs demonstrating apelin-like immunoreactivity (apelin-LI) in the human heart. Apelin-LI is expressed in vascular endothelial cells of right atria (a and b) and left ventricle (c and e), with adjacent sections (d and f) stained for the endothelial marker von Willebrand factor (vWF). Apelin-LI was also identified in endocardial endothelial cells lining the right atrium (g) with an adjacent section stained for vWF (h). In sections from right atrium (i) no staining was detectable when the primary antiserum was omitted as negative control (j).

Kleinz et al. Regul Pept. 2004 May 15;118(3):119-25.

 

Photomicrographs showing apelin-like immunoreactivity in vascular endothelial cells

fig04

Photomicrographs showing apelin-like immunoreactivity in vascular endothelial cells of blood vessels from kidney (a, b) and adrenal gland (c) with an adjacent section of adrenal gland (d), where the primary antiserum was omitted as negative control.

Kleinz et al. Regul Pept. 2004 May 15;118(3):119-25.

 

fig05

Apelin-like immunoreactivity in vascular endothelial cells of large conduit vessels. Photomicrographs show staining in saphenous vein (a) and coronary artery (c) with higher magnification of the respective vessels in (b) and (d).

Kleinz et al. Regul Pept. 2004 May 15;118(3):119-25.

 

Apelin-36 Antibody Immunoblot Analyses (G-057-15).

apelin western blot analysis

Figure 1. Apelin-36 Antibody Immunoblot Analyses. Immunoblot analysis of whole cell lysates using the apelin-36 antibody (from Phoenix Pharmaceuticals). (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 IAA 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.

Lee et al. Endocrinology. 2005 Jan;146(1):231-6.

 

Apelin-36 (Human) - Magnetic Bead RIA kit (MRK-057-15)

Only 5 to 40 µL of sample are necessary for accurate peptide level determination using Phoenix’s Magnetic Bead RIA kits. Please contact us at support@phoenixpeptide.com for more information on this product.

 

Apelin-36 (Human) EIA Kit (EK-057-15) and Fluorescent EIA Kit (FEK-057-15)

Linear Range: 0.08-1.48 ng/ml
Extraction Free

Linear Range: 20-480 pg/ml
4 times more sensitive than normal EIA kits

Apelin-12 (Human, Rat, Mouse, Bovine) EIA Kit (EK-057-23) and Fluorescent EIA Kit (FEK-057-23)

Linear Range: 0.07-0.79 ng/ml

Linear Range: 15.8-419 pg/ml
4 times more sensitive than normal EIA kits

Apelin-36 (Rat, Mouse) EIA Kit (EK-057-28), Fluorescent EIA Kit (FEK-057-28) and Chemiluminescent EIA Kit (CEK-057-28)

Linear Range: 0.26-2.0 ng/ml
Extraction Free

Linear Range: 5.7 - 346 pg/ml
45 times more sensitive than normal EIA kits

Linear Range: 12.7 - 126 pg/ml
20 times more sensitive than normal EIA kits

 

Apelin-36 (Human) (RK-057-15) and Apelin-36 (Rat, Mouse) (RK-057-28) RIA Kit

RK-057-15 Apelin-36 (Human) RIA Kit

 

Amino acid sequences of apelin-13 (a) and apelin-36

apelin-13

Amino acid sequences of apelin-13 (a) and apelin-36 (b). Dark grey indicates amino acids identical in both peptides. The N-terminal Pyr1 of (Pyr1) apelin-13 differs from the prepropeptide sequence as a result of post-translational modification. The antiserum used for immunocytochemistry was raised against the C-terminal dodecapeptide common to both peptides, which is indicated in dark grey.

Kleinz et al. Regul Pept. 2004 May 15;118(3):119-25.

 

Prepro-Apelin (Human) Prohormone Schematic

 

Amino Acid Sequence of Human, Mouse, Rat & Bovine Prepro Apelin

 

Sequence of apelin analogues and related peptides

Apelin

 

Genomic Structure of the Human Proapelin Gene

A: An alignment of amino acid sequences of rat preproapelin with human and bovine preproapelin. Conserved amino acids are shown boxed. The mature apelin peptide is shaded. Numeric amino acid positions are indicated on the right. The GenBank accession numbers for rat and human preproapelin are AF179679 and AF179680, respectively.

B: An alignment of amino acid sequences of human apelin and angiotensin II. Conserved amino acids are shown boxed. C: The genomic structure of the human preproapelin gene as found in the human PAC 454M7 clone (GenBank accession no. AL022162). Nucleotide positions of PAC 454M7 defining preproapelin gene exons (boxes) are shown at the top. The ORF is shown in black, with the nucleotide positions of the start and stop codons shown at bottom.

Lee et al. J Neurochem. 2000 Jan;74(1):34-41.

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APJ_Receptor;SCNH2

%apelin%


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