Phoenix Pharmaceuticals, Inc.

Apelin
A Cardiovascular Peptide related to Heart Failure

A new apelin receptor antagonist is available for your research!

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.

Immunocytochemical localization of the endogenous vasoactive peptide apelin to human vascular and endocardial endothelial cells

Apelin, the proposed endogenous peptide ligand of the novel G-protein-coupled receptor APJ, has been shown to possess potent vasodilator and positive inotropic effects in rats and humans in vivo. However, in humans, no endogenous source of apelin has been reported. Therefore, based on the presence of APJ and mRNA encoding apelin in human tissues, we investigated the expression of apelin in freshfrozen human tissue from right atrium, left ventricle, lung, kidney, adrenal and large conduit vessels using immunocytochemistry. Apelin-like immunoreactivity (apelin-LI) was detected in vascular endothelial cells lining blood vessels in the human heart, kidney, adrenal gland and lung and in endothelial cells of large conduit vessels. Apelin-LI was also present in endocardial endothelial cells lining recesses of the right atrium. Apelin-LI was not present or below the level of detection in cardiomyocytes, Purkinje’s cells, pulmonary or renal epithelial cells, secretory cells of the adrenal gland, vascular smooth muscle cells, adipocytes, nerves and connective tissue. The restricted presence of apelin- LI in endothelial cells suggests that endothelial apelin may play a role as a locally secreted cardiovascular mediator acting on APJ receptors present on the vascular smooth muscle and on cardiac myocytes to regulate vascular tone and cardiac contractility.
Matthias J. Kleinz*, Anthony P. Davenport. Regulatory Peptides 118 (2004) 119–125

Novel role for the potent endogenous inotrope apelin in human cardiac dysfunction
BACKGROUND: Apelin is among the most potent stimulators of cardiac contractility known. However, no physiological or pathological role for apelin-angiotensin receptor-like 1 (APJ) signaling has ever been described. METHODS AND RESULTS: We performed transcriptional profiling using a spotted cDNA microarray with 12 814 unique clones on paired samples of left ventricle obtained before and after placement of a left ventricular assist device in 11 patients. The significance analysis of microarrays and a novel rank consistency score designed to exploit the paired structure of the data confirmed that natriuretic peptides were among the most significantly downregulated genes after offloading. The most significantly upregulated gene was the G-protein-coupled receptor APJ, the specific receptor for apelin. We demonstrate here using immunoassay and immunohistochemical techniques that apelin is localized primarily in the endothelium of the coronary arteries and is found at a higher concentration in cardiac tissue after mechanical offloading. These findings imply an important paracrine signaling pathway in the heart. We additionally extend the clinical significance of this work by reporting for the first time circulating human apelin levels and demonstrating increases in the plasma level of apelin in patients with left ventricular dysfunction. CONCLUSIONS: The apelin-APJ signaling pathway emerges as an important novel mediator of cardiovascular control.
Chen MM, et al. Circulation. 2003 Sep 23;108(12):1432-9. Epub 2003 Sep 08

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

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 staurosporine 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 I, 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 K, 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 GC, Dun SL, Yang J, Ohsawa M, Chang JK, Dun NJ. Neurosci Lett 2002 Jul 26;327(3):193-7

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.

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

Matthias J. Kleinz*, Anthony P. Davenport. Regulatory Peptides 118 (2004) 119–125

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). M.J. Kleinz, A.P. Davenport / Regulatory Peptides 118 (2004) 119–125 121

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. Matthias J. Kleinz*, Anthony P. Davenport. Regulatory Peptides 118 (2004) 119–125

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). Matthias J. Kleinz*, Anthony P. Davenport. Regulatory Peptides 118 (2004) 119–125

Specificity of the rabbit-anti-apelin-12 antiserum determined by antiserum dilution ELISA. Microtiter plates were coated with a fixed amount (1 Ag/ml) of five different peptides, apelin-36 (o), apelin-13 (.), ghrelin (B), ET-1 (4), angiotensin-II (5). The antiserum cross-reacted with apelin-36 and apelin-13, but not with the other peptides. Absorbance (meanFS.E.M.) is plotted against antiserum dilution as a measure of potency and specificity of the antibody–antigen interaction. Matthias J. Kleinz*, Anthony P. Davenport. Regulatory Peptides 118 (2004) 119–125

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. Matthias J. Kleinz*, Anthony P. Davenport. Regulatory Peptides 118 (2004) 119–125

Catalog Number Product Name Quantity $US/Euro Order 057-28 Apelin-36 (Rat) 100 µg 150         EK-057-28 Apelin-36 (Rat) - EIA Kit 96 wells 450
Apelin Receptor APJ Immunohistochemistry

Apelin level and distribution in human left ventricle. A, Left ventricular tissue apelin level as determined by enzyme immunoassay. The level rose significantly (p<0.001) after offloading by implantation of a left ventricular assist device. Units are nanogram per milliliter. B, Immunohistochemical distribution of apelin. Apelin, labeled reddish-brown, is highly localized to endothelial and smooth muscle cells in diseased (right) and normal heart (diseased only shown). Staining of consecutive sections with platelet-endothelial cell adhesion molecule (CD31, middle) confirms the specificity of this localization. Control panels (left) represent sections where incubation in primary antibody was omitted. Chen MM, et al. Circulation. 2003 Sep 23;108(12):1432-9. Epub 2003 Sep 08
Plasma apelin levels in heart failure. A. There were significant increases in the plasma level of apelin as determined by enzyme immunoassay in early heart failure through NYHA class 2 (p<0.02). In later stage diseases, the mean level id lower, although this change is not significant. Class 4 patients (n=7) are combined with class 3 (from left to right, n=34, 24, 12, and 38). B, Apelin rises in mild-to-moderate left ventricular dysfunction but falls in severe disease (p<0.02 for both). Normal is defined as a left ventricular ejection fraction greater than 45%, mild to moderate is 25% to 45% and severe is less than 25% (from left to right, n=42, 28, 40). Chen MM, et al. Circulation. 2003 Sep 23;108(12):1432-9. Epub 2003 Sep 08

Foldes G, et al. Biochem Biophys Res Commun. 2003 Aug 29;308(3):480-5
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 °C and kept at -80 °C 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 l of 40% acetonitrile in aqueous 0.1% TFA. Plasma samples (1 ml) were extracted with SepPak C18 cartridges, dried, and reconstituted in 400 l 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).
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 ?nbsp;145 pg/mg, n=5 and 2.8 ?nbsp;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 ?nbsp;0.5 pg/mg, p=0.07) and idiopathic dilated cardiomyopathy (3.8 ?nbsp;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 ?nbsp;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 ?nbsp;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 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. Foldes G, et al. Biochem Biophys Res Commun. 2003 Aug 29;308(3):480-5	Fig.02 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. Foldes G, et al. Biochem Biophys Res Commun. 2003 Aug 29;308(3):480-5
Fig. 3. 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. Foldes G, et al. Biochem Biophys Res Commun. 2003 Aug 29;308(3):480-5
IDC, idiopathic dilated cardiomyopathy; CCHD, coronary heart disease. Foldes G, et al. Biochem Biophys Res Commun. 2003 Aug 29;308(3):480-5
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, Dennis K., et al. Characterization of Apelin, the Ligand for the APJ Receptor. Journal of Neurochemistry  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 g of poly(A)+ RNA isolated from various tissues, with the exception of rat pituitary (7.5 g), olfactory tubercle (7.8 g), septum (5 g), and liver (7 g). Lee, Dennis K., et al. Characterization of Apelin, the Ligand for the APJ Receptor. Journal of Neurochemistry  74 (1), 34-41.
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. Lee, Dennis K., et al. Characterization of Apelin, the Ligand for the APJ Receptor. Journal of Neurochemistry  74 (1), 34-41.
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. Lee, Dennis K., et al. Characterization of Apelin, the Ligand for the APJ Receptor. Journal of Neurochemistry  74 (1), 34-41.
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. Lee, Dennis K., et al. Characterization of Apelin, the Ligand for the APJ Receptor. Journal of Neurochemistry  74 (1), 34-41.
Water consumption after intraperitoneal injection of 100 g 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.
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)	Reference: Neurosci Lett 2002 Jul 26;327(3):193-7

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

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

Prepro-Apelin (Human) Sequence

	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, S.D. et al. Br. J. Pharmacol. 132(6), 1255-1260 (2001)

New Apelin-12 EIA Kit is available for cardiovascular research!!!

Specificity   Peptide % Crossreactivity Apelin-12 (Human) 100 Apelin-13 (Human) 100 Apelin-36 (Human) 100 Apelin-36 (Rat) 100

Specificity   Peptide % Crossreactivity Apelin-36 (Human) 100 Apelin-36 (Rat) 100 Apelin-16 (Human) 0.10 Apelin-13 (Human) 0.27

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