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 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.     

Apelin protects myocardial injury induced by isoproterenol in rats

We aimed to explore the change in level of apelin and its receptor APJ during myocardial injury and the therapeutic effects of apelin in myocardial injury. Rat myocardial injury was induced by subcutaneous injection of a high dose of isoproterenol (ISO); apelin and APJ mRNA levels were determined by RT-PCR; APJ protein was determined by Western blot; EIA and RIA were used to measure the apelin content and receptor binding, respectively. Plasma lactate dehydrogenase (LDH) activity and myocardial and plasma malondialdehyde (MDA) contents were higher in ISO-treated hearts than that in controls. ISO-treated rats showed lower +/-LV dp/dt(max) values and higher LVEDP value (all P<0.01), which suggested severe heart failure. As well, the apelin content in plasma, atrial and ventricular myocardium was decreased by 27%, 30% and 25% (P<0.01), respectively. The mRNA levels of apelin and APJ in myocardia were also markedly reduced; but the APJ protein level in myocardia was increased. However, administration of apelin significantly ameliorated myocardial injury and ISO-induced heart failure. Compared with the ISO-alone group, the group given low-dosage apelin (5 nmol/kg/day) had 39% and 66% higher +LV dp/dt(max) and -LV dp/dt(max) values, and 40.7% lower LVEDP value (P<0.01), and the leakage of myocardial LDH and increased MDA content were attenuated (all P<0.01). Interestingly, bolus injections of apelin (10 nmol/kg/day) resulted in potent inotropic effects in ISO-treated rats. ISO-induced myocardial injury resulted in hypoexpression of apelin and its receptor APJ, and the administration of exogenous apelin ameliorated heart failure and myocardial injury. Apelin could have a cardioprotective effect, and the apelin-APJ system may be a new therapeutic target in myocardial injury and heart failure.     

Effect of Intracerebroventricular Administration of Apelin-13 on the Hypothalamus–Pituitary–Thyroid Axis and Peripheral Uncoupling Proteins

Apelin, a ligand for G protein-coupled APJ receptor, is a peptide hormone. Although apelin and APJ receptors are determined in hypothalamus and thyroid gland its role in the hypothalamus–pituitary–thyroid (HPT) axis and mechanism of action on energy metabolism is not clear. This suggests that apelin may play a role in the HPT axis and energy metabolism. This study was designed to determine possible effects of centrally administered apelin-13 on the HPT axis and energy metabolism. A total of 40 adult male Sprague Dawley rats were divided into four groups (n?=?10 each group). Intact rats served as control group while the sham group received vehicle of apelin. Apelin-13 was injected intracerebroventricularly at the doses of 1 and 10 nmol, for 7 days in the rats in the experimental group. At the end of the experimental protocol, animals were decapitated and brain, blood, white and brown adipose tissues samples were collected. There was no significant difference between the groups in terms of hypothalamic TRH mRNA levels. Serum TSH levels were significantly higher in all groups compared to the control group (p?<?0.05). Serum fT3 and fT4 levels were significantly lower in apelin-13 administered groups (p?<?0.05). Moreover, apelin-13 administered groups had lower levels of UCP1 mRNA in white and brown adipose tissues. UCP3 mRNA expression in muscle tissue was also lower in apelin-13 treated groups (p?<?0.05). These results indicates that apelin-13 exhibits a decreasing effect on energy consumption through a mechanism involving the peripheral rather than central arms of the HPT axis.     

Apelin-13 limits infarct size and improves cardiac postischemic mechanical recovery only if given after ischemia

We studied whether apelin-13 is cardioprotective against ischemia/reperfusion injury if given as either a pre- or postconditioning mimetic and whether the improved postischemic mechanical recovery induced by apelin-13 depends only on the reduced infarct size or also on a recovery of function of the viable myocardium. We also studied whether nitric oxide (NO) is involved in apelin-induced protection and whether the reported ischemia-induced overexpression of the apelin receptor (APJ) plays a role in cardioprotection. Langendorff-perfused rat hearts underwent 30 min of global ischemia and 120 min of reperfusion. Left ventricular pressure was recorded. Infarct size and lactate dehydrogenase release were determined to evaluate the severity of myocardial injury. Apelin-13 was infused at 0.5 μM concentration for 20 min either before ischemia or in early reperfusion, without and with NO synthase inhibition by N(G)-nitro-l-arginine (l-NNA). In additional experiments, before ischemia also 1 μM apelin-13 was tested. APJ protein level was measured before and after ischemia. Whereas before ischemia apelin-13 (0.5 and 1.0 μM) was ineffective, after ischemia it reduced infarct size from 54 ± 2% to 26 ± 4% of risk area (P < 0.001) and limited the postischemic myocardial contracture (P < 0.001). l-NNA alone increased postischemic myocardial contracture. This increase was attenuated by apelin-13, which, however, was unable to reduce infarct size. Ischemia increased APJ protein level after 15-min perfusion, i.e., after most of reperfusion injury has occurred. Apelin-13 protects the heart only if given after ischemia. In this protection NO plays an important role. Apelin-13 efficiency as postconditioning mimetic cannot be explained by the increased APJ level.     

Apelin-36, a potent peptide,protects against ischemic brain injury by activating the PI3K/Akt pathway

Apelin is an endogenous ligand of G protein-coupled receptor-apelin and angiotensin-1-like receptor (APJ). The biological effects of apelin–APJ system are reported in multiple systems including cardiovascular, endocrinal, and gastrointestinal system. Previous studies had shown that apelin-13 is a potential protective agent on cardiac ischemia; however, the role of apelin in the central nervous system remained unknown. In this study, we investigated therapeutic effects of apelin-36, a long form of apelin, in ischemic brain injury models. We found that apelin-36 reduced cerebral infarct volume in the middle cerebral artery occlusion (MCAO) model and the neonatal hypoxic/ischemic (H/I) injury model. Apelin-36 improved neurological deficits in the MCAO model and promoted long-term functional recovery after H/I brain injury. We further explored the protective mechanisms of apelin-36 on H/I brain injury. We clearly demonstrated that apelin-36 significantly reduced the levels of cleaved caspase-3 and Bax, two well-established apoptotic markers after H/I injury, indicating the anti-apoptotic activity of apelin-36 in ischemic injury. Since apelin-36 increased the level of phosphorylated Akt after H/I injury, we treated neonates with a specific PI3K inhibitor LY294002. We found that LY294002 decreased the phosphorylated Akt level and attenuated protective effects of apelin-36 on apoptosis. These suggested that the PI3K/Akt pathway was at least in part involved in the anti-apoptotic mechanisms of apelin-36. Our findings demonstrated that apelin-36 was a promising therapeutic agent on the treatment of ischemic brain injury.     

Central and peripheral cardiovascular actions of apelin in conscious rats

APJ was cloned as an orphan G protein-coupled receptor and shares a close identity with angiotensin II type 1 receptor (AT1R). Apelin is a peptide that has recently been identified as an endogenous ligand of the APJ. Apelin and APJ mRNA are expressed in peripheral tissue and the central nervous system. However, little is known about the effects of apelin in cardiovascular regulation. To examine the central and peripheral role of apelin, we injected the active fragment of apelin [(Pyr1)apelin-13] intracerebroventricularly (ICV, 5 and 20 nmol, n=6) or intravenously (IV, 20 and 50 nmol, n=4 or 5) in conscious rats. ICV injection of (Pyr1)apelin-13 dose-dependently increased mean arterial pressure (MAP) and heart rate (HR) (19+/-3 mm Hg and 162+/-26 bpm at 20 nmol). Pretreatment with ICV injection of the AT1R antagonist (CV-11974, 20 nmol) did not alter the apelin-induced increase in MAP and HR. IV injection of (Pyr1)apelin-13 also dose-dependently increased MAP and HR (13+/-2 mm Hg and 103+/-18 bpm at 50 nmol); however, the peripheral effects of apelin were relatively weak compared to its central effects. Expression of c-fos in the paraventricular nucleus (PVN) of hypothalamus was increased in the rat that received ICV injection of (Pyr1)apelin-13 but not in the rat that received IV injection of (Pyr1)apelin-13. These results suggest that apelin plays a role in both central and peripheral cardiovascular regulation in conscious rats, and that the cardiovascular effects of apelin are not mediated by the AT1R.     

Apelin reduces myocardial reperfusion injury independently of PI3K/Akt and P70S6 kinase

Apelin, the endogenous ligand of the G protein-coupled APJ receptor, is a peptide mediator with emerging regulatory actions in the heart. The aim of the present studies was to explore potential roles of the apelin/APJ system in myocardial ischaemia/reperfusion injury. To determine the cardiac expression of apelin/APJ and potential regulation by acute ischaemic insult, Langendorff perfused rat hearts were subjected to regional ischaemia (left coronary artery occlusion, 35 min) or ischaemia followed by reperfusion (30 min). Apelin and APJ mRNA expression were then determined in ventricular myocardium by rt-PCR. Unlike APJ mRNA expression, which remained unchanged, apelin mRNA was upregulated 2.4 fold in ventricular myocardium from isolated rat hearts undergoing ischaemia alone, but returned back to control levels after 30 min reperfusion. We then proceeded to test the hypothesis that treatment with exogenous apelin is protective against ischaemia/reperfusion injury. Perfused hearts were subjected to 35 min left main coronary artery occlusion and 120 min reperfusion, after which infarct size was determined by tetrazolium staining. Exogenous Pyr(1)-apelin-13 (10(-8 )M) was perfused either from 5 min prior to 15 min after coronary occlusion, or from 5 min prior to 15 min after reperfusion. Whilst ineffective when used during ischaemia alone, apelin administered during reperfusion significantly reduced infarct size (47.6+/-2.6% of ischaemic risk zone compared to 62.6+/-2.8% in control, n=10 each, p<0.05) in hearts subject to temporary coronary occlusion followed by reperfusion. This protective effect was not abolished by co-administration of the PI3K inhibitor wortmannin (10(-7 )M, infarct size 49.8+/-4.1%, n=4) or the P70S6 kinase inhibitor rapamycin (10(-9 )M, 41.8+/-8.8%, n=4). In conclusion these results suggest that apelin may be a new and potentially important cardioprotective autacoid, upregulated rapidly after myocardial ischaemia and acting through an unknown pathway.     

Apelin/APJ system: A potential therapeutic target for endothelial dysfunction-related diseases

APJ is a G protein-coupled receptor and its endogenous ligand is apelin. Studies have shown that apelin/APJ system is widely distributed in the body, especially highly expressed in the vascular endothelial cells (ECs). Numerous reports have demonstrated that apelin/APJ system plays an important role in the regulation of ECs function. Our lab has demonstrated that apelin-13 is able to promote adhesion of monocyte-human umbilical vein EC via 14-3-3, and reactive oxygen species-autophagy signaling pathways. In this review, we concentrate on the regulatory mechanism of apelin/APJ system in EC, including promotion of proliferation, migration, and angiogenesis. Moreover, we also analyze the role of apelin/APJ on endothelial dysfunction-related diseases including atherosclerosis, diabetes, hypertension, and myocardial infarction. Finally, we summarize the most commonly used agonists and antagonists of APJ. Therefore, apelin/APJ system is expected to be a therapeutic target for the treatment of endothelial dysfunction-related diseases.     

In vivo genetic profiling and cellular localization of apelin reveals a hypoxia-sensitive, endothelial-centered pathway activated in ischemic heart failure

Signaling by the peptide ligand apelin and its cognate G protein-coupled receptor APJ has a potent inotropic effect on cardiac contractility and modulates systemic vascular resistance through nitric oxide-dependent signaling. In addition, there is evidence for counterregulation of the angiotensin and vasopressin pathways. Regulatory stimuli of the apelin-APJ pathway are of obvious importance but remain to be elucidated. To better understand the physiological response of apelin-APJ to disease states such as heart failure and to elucidate the mechanism by which such a response might occur, we have used the murine model of left anterior descending coronary artery ligation-induced ischemic cardiac failure. To identify the key cells responsible for modulation and production of apelin in vivo, we have created a novel apelin-lacZ reporter mouse. Data from these studies demonstrate that apelin and APJ are upregulated in the heart and skeletal muscle following myocardial injury and suggest that apelin expression remains restricted to the endothelium. In cardiac failure, endothelial apelin expression correlates with other hypoxia-responsive genes, and in healthy animals both apelin and APJ are markedly upregulated in various tissues following systemic hypoxic exposure. Experiments with cultured endothelial cells in vitro show apelin mRNA and protein levels to be increased by hypoxia, through a hypoxia-inducible factor-mediated pathway. These studies suggest that apelin-expressing endothelial cells respond to conditions associated with heart failure, possibly including local tissue hypoxia, and modulate apelin-APJ expression to regulate cardiovascular homeostasis. The apelin-APJ pathway may thus provide a mechanism for systemic endothelial monitoring of tissue perfusion and adaptive regulation of cardiovascular function.     

Apelin-13 increases myocardial progenitor cells and improves repair postmyocardial infarction

Apelin is an endogenous ligand for the angiotensin-like 1 receptor (APJ) and has beneficial effects against myocardial ischemia-reperfusion injury. Little is known about the role of apelin in the homing of vascular progenitor cells (PCs) and cardiac functional recovery postmyocardial infarction (post-MI). The present study investigated whether apelin affects PC homing to the infarcted myocardium, thereby mediating repair and functional recovery post-MI. Mice were infarcted by coronary artery ligation, and apelin-13 (1 mg·kg(-1)·day(-1)) was injected for 3 days before MI and for 14 days post-MI. Homing of vascular PCs [CD133(+)/c-Kit(+)/Sca1(+), CD133(+)/stromal cell-derived factor (SDF)-1α(+), and CD133(+)/CXC chemokine receptor (CXCR)-4(+)] into the ischemic area was examined. Myocardial Akt, endothelial nitric oxide synthase (eNOS), VEGF, jagged1, notch3, SDF-1α, and CXCR-4 expression were assessed at 24 h and 14 days post-MI. Functional analyses were performed on day 14 post-MI. Mice that received apelin-13 treatment demonstrated upregulation of SDF-1α/CXCR-4 expression and dramatically increased the number of CD133(+)/c-Kit(+)/Sca1(+), CD133(+)/SDF-1α(+), and c-Kit(+)/CXCR-4(+) cells in infarcted hearts. Apelin-13 also significantly increased Akt and eNOS phosphorylation and upregulated VEGF, jagged1, and notch3 expression in ischemic hearts. This was accompanied by a significant reduction of myocardial apoptosis. Furthermore, treatment with apelin-13 promoted myocardial angiogenesis and attenuated cardiac fibrosis and hypertrophy together with a significant improvement of cardiac function at 14 days post-MI. Apelin-13 increases angiogenesis and improves cardiac repair post-MI by a mechanism involving the upregulation of SDF-1α/CXCR-4 and homing of vascular PCs.     

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, [相似文献   

Apelin-13 Enhances Arcuate POMC Neuron Activity via Inhibiting M-Current

The hypothalamus is a key element of the neural circuits that control energy homeostasis. Specific neuronal populations within the hypothalamus are sensitive to a variety of homeostatic indicators such as circulating nutrient levels and hormones that signal circulating glucose and body fat content. Central injection of apelin secreted by adipose tissues regulates feeding and glucose homeostasis. However, the precise neuronal populations and cellular mechanisms involved in these physiological processes remain unclear. Here we examine the electrophysiological impact of apelin-13 on proopiomelanocortin (POMC) neuron activity. Approximately half of POMC neurons examined respond to apelin-13. Apelin-13 causes a dose-dependent depolarization. This effect is abolished by the apelin (APJ) receptor antagonist. POMC neurons from animals pre-treated with pertussis toxin still respond to apelin, whereas the Gβγ signaling inhibitor gallein blocks apelin-mediated depolarization. In addition, the effect of apelin is inhibited by the phospholipase C and protein kinase inhibitors. Furthermore, single-cell qPCR analysis shows that POMC neurons express the APJ receptor, PLC-β isoforms, and KCNQ subunits (2, 3 and 5) which contribute to M-type current. Apelin-13 inhibits M-current that is blocked by the KCNQ channel inhibitor. Therefore, our present data indicate that apelin activates APJ receptors, and the resultant dissociation of the Gαq heterotrimer triggers a Gβγ-dependent activation of PLC-β signaling that inhibits M-current.     

Unaltered ryanodine receptor protein levels in ischemic cardiomvopathy

Previous studies on sarcoplasmic reticulum calcium release channel (ryanodine receptor) demonstrated that protein levels are unchanged in myocardium from hearts with end-stage failing dilated cardiomyopathy. In ischemic cardiomyopathy, ryanodine receptor mRNA levels were shown to be decreased but no data on protein levels are available. Accordingly, protein levels of ryanodine receptor, calsequestrin, and sarcoplasmic reticulum calcium-ATPase (SR-Ca²⁺-ATPase) were measured by Western blot analysis in nonfailing human myocardium (n = 7) and in end-stage failing myocardium due to ischemic cardiomyopathy (n = 14). Protein levels of calsequestrin which is the major sarcoplasmic reticulum calcium storage protein were similar in nonfailing myocardium and in myocardium from end-stage failing hearts with ischemic cardiomyopathy. Ryanodine receptor protein levels, normalized to total protein or calsequestrin were also unchanged in ischemic cardiomyopathy. In contrast, protein levels of SR-Ca²⁺-ATPase normalized to total protein or calsequestrin were decreased by 31 and 30%, respectively (p < 0.05). The data indicate that (I) sarcoplasmic reticulum calcium uptake sites are decreased relative to the release sites in ischemic cardiomyopathy, and (2) alterations of sarcoplasmic proteins are similar in ischemic and dilated cardiomyopathy.     

Cardiovascular effects of a PEGylated apelin

Several studies have documented cardiovascular effects of apelin, including enhanced inotropy and vasodilation. However, these cardiovascular effects are short lived due to the predicted short circulating half-life of the apelin peptide. To address this limitation of apelin, we pursued N-terminal PEGylation of apelin and examined the cardiovascular effects of the PEGylated apelin. A 40 kDa PEG conjugated apelin-36 (PEG–apelin-36) was successfully produced with N-terminal conjugation, high purity (>98%) and minimum reduction of APJ receptor binding affinity. Using an adenylate cyclase inhibition assay, comparable in vitro bioactivity was observed between the PEG–apelin-36 and unmodified apelin-36. In vivo evaluation of the PEG–apelin-36 was performed in normal rats and rats with myocardial infarction (MI). Cardiac function was assessed via echocardiography before, during a 20 min IV infusion and up to 100 min post peptide infusion. Similar increases in cardiac ejection fraction (EF) were observed during the infusion of PEG–apelin-36 and apelin-36 in normal rats. However, animals that received PEG–apelin-36 maintained significantly increased EF over the 100 min post infusion monitoring period compared to the animals that received unmodified apelin-36. Interestingly, EF increases observed with PEG–apelin-36 and apelin-36 were greater in the MI rats. PEG–apelin-36 had a prolonged circulating life compared to apelin-36 in rats. There were no changes in aortic blood pressure when PEG–apelin-36 or apelin-36 was administered. To our knowledge this is the first report of apelin PEGylation and documentation of its cardiovascular effects.     

Targeting drugs to APJ receptor: From signaling to pathophysiological effects

G-protein-coupled receptors (GPCRs) are recognized as the largest protein receptor superfamily, which are widely distributed in various tissues and organs. In addition, GPCRs are involved in many physiological and pathological longitudinal responses. Studies have indicated that putative receptor protein related to AT1 (APJ receptor) is an orphan GPCRs until its endogenous ligand apelin is found. Recently, Elabela, a new APJ receptor endogenous ligand was also found. Some evidence showed that the APJ receptor is distributed in the central nervous and cardiovascular systems. Moreover, the APJ receptor and its ligand are involved in many physiological functions and pathophysiological effects, making it a promising drug target for future treatment of diseases such as ischemic heart disease, hypertension, heart failure, and others. Although APJ is closely associated with many diseases, there are no drugs that can activate or inhibit APJ directly. In the current review, we try our best to summarize all agonists and antagonists targeting APJ, including peptides and small molecules. Given the role of apelin/APJ and Elabela/APJ in cardiovascular and other diseases, we believe that the combination of these agonists and antagonists with apelin and Elabela will play a corresponding role in various pathophysiological effects with further development of research.     

Apelin-13 induces ERK1/2 but not p38 MAPK activation through coupling of the human apelin receptor to the Gi2 pathway

Apelin signaling to the family of mitogen-activated protein kinases (MAPKs), such as extracellular-regulated kinases 1/2 (ERK1/2) and p38 MAPK, through the coupling of apelin receptor (APJ) to G-protein, mediates important pathophysiological responses. Although apelin fragments have been reported to induce ERK1/2 activation through G_i-protein, the intracellular pathways by which APJ activates these MAPKs are only partially understood. Here, using stably transfected human embryonic kidney 293 (HEK293) cells overexpressing human APJ (HEK293-apelinR), we showed that apelin-13 signaling leads to ERK1/2 and p38 MAPK pathways through APJ activation. It was found in HEK293-apelinR cells that ERK1/2 activation was initiated by apelin-13 at 5 min, with the peak of activation occurring at 15 min, and a return to the basal level within 60 min. The activation of ERK1/2 appeared to be dose-dependent with a significant activation being observed at 10 nM apelin-13 and maximal activation at 100 nM. However, phosphorylated-p38 MAPK was not detected in HEK293-apelinR cells treated with apelin-13. We also shown that the apelin-13-induced ERK1/2 activation requires a coupling with pertussis toxin-sensitive G-protein, and that overexpression of dominant-negative G_i2 completely inhibits the apelin-13-induced ERK1/2 activation. In addition, treatment with apelin-13 resulted in a concentration-dependent reduction of forskolin-stimulated cAMP production. It is therefore suggested that apelin-13 activates ERK1/2 but not p38 MAPK, which involves the coupling of APJ to the G_i2 cascade. In conclusion, the ERK1/ 2, but not p38 MAPKpathway is activated by apelin-13 through coupling of human APJ to G_i2-protein, which contributes to cellular responses.     

Structural and functional study of the apelin-13 peptide,an endogenous ligand of the HIV-1 coreceptor,APJ

The APJ receptor is widely expressed in the human central nervous system (CNS). Apelin was recently identified as the endogenous peptidic ligand for human APJ. Studies with animal models suggested that APJ and apelin play an important role in the hypothalamic regulation of water intake and the endocrine axis, in the regulation of blood pressure, and in cardiac contractility. Apelin has been found to block the activity of APJ as a human immunodeficiency virus type I (HIV-1) coreceptor. In this study, we combined chemical synthetic approaches with alanine substitution to evaluate the structural requirements for interactions with the APJ receptor. We demonstrated that apelin peptides in aqueous solution adopt a random conformation, and the positive charge and hydrophobic residues of apelin-13 play important roles in interactions with the APJ receptor. We have observed an important correlation between receptor binding affinity and cell-cell fusion inhibitory activity. The elucidation of structural requirements of apelin-13 in its interaction with the APJ receptor is critical for further investigation of apelin-APJ functions in vivo and in the design of small molecular inhibitors for potential treatment of HIV-1 infection in the CNS.     

Identification of Serine 348 on the Apelin Receptor as a Novel Regulatory Phosphorylation Site in Apelin-13-induced G Protein-independent Biased Signaling

Phosphorylation plays vital roles in the regulation of G protein-coupled receptor (GPCR) functions. The apelin and apelin receptor (APJ) system is involved in the regulation of cardiovascular function and central control of body homeostasis. Here, using tandem mass spectrometry, we first identified phosphorylated serine residues in the C terminus of APJ. To determine the role of phosphorylation sites in APJ-mediated G protein-dependent and -independent signaling and function, we induced a mutation in the C-terminal serine residues and examined their effects on the interaction between APJ with G protein or GRK/β-arrestin and their downstream signaling. Mutation of serine 348 led to an elimination of both GRK and β-arrestin recruitment to APJ induced by apelin-13. Moreover, APJ internalization and G protein-independent ERK signaling were also abolished by point mutation at serine 348. In contrast, this mutant at serine residues had no demonstrable impact on apelin-13-induced G protein activation and its intracellular signaling. These findings suggest that mutation of serine 348 resulted in inactive GRK/β-arrestin. However, there was no change in the active G protein thus, APJ conformation was biased. These results provide important information on the molecular interplay and impact of the APJ function, which may be extrapolated to design novel drugs for cardiac hypertrophy based on this biased signal pathway.     

The apelinergic system in the developing lung: expression and signaling

Apelin and its receptor APJ constitute a signaling pathway best recognized as an important regulator of cardiovascular homeostasis. This multifunctional peptidergic system is currently being described to be involved in embryonic events which extend into vascular, ocular and heart development. Additionally, it is highly expressed in pulmonary tissue. Therefore, the aim of this study was to investigate the role of apelinergic system during fetal lung development. Immunohistochemistry and Western blot analysis were used to characterize apelin and APJ expression levels and cellular localization in normal fetal rat lungs, at five different gestational ages as well as in the adult. Fetal rat lung explants were cultured in vitro with increasing doses of apelin. Treated lung explants were morphometrically analyzed and assessed for MAPK signaling modifications. Both components of the apelinergic system are constitutively expressed in the developing lung, with APJ exhibiting monomeric, dimeric and oligomeric forms in the pulmonary tissue. Pulmonary epithelium also displayed constitutive nuclear localization of the receptor. Fetal apelin expression is higher than adult expression. Apelin supplementation inhibitory effect on branching morphogenesis was associated with a dose dependent decrease in p38 and JNK phosphorylation. The results presented provide the first evidence of the presence of an apelinergic system operating in the developing lung. Our findings also suggest that apelin inhibits fetal lung growth by suppressing p38 and JNK signaling pathways.