This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 91/5/434    most recent 01.RES.0000033522.37861.69v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Szokodi, I. Articles by Ruskoaho, H. Search for Related Content PubMed PubMed Citation Articles by Szokodi, I. Articles by Ruskoaho, H. Related Collections Contractile function Receptor pharmacology

(Circulation Research. 2002;91:434.)
© 2002 American Heart Association, Inc.

Integrative Physiology

Apelin, the Novel Endogenous Ligand of the Orphan Receptor APJ, Regulates Cardiac Contractility

István Szokodi, Pasi Tavi, Gábor Földes, Sari Voutilainen-Myllylä, Mika Ilves, Heikki Tokola, Sampsa Pikkarainen, Jarkko Piuhola, Jaana Rysä, Miklós Tóth, Heikki Ruskoaho

From the Departments of Pharmacology and Toxicology (I.Sz., H.T., S.P., J.P., J.R., H.R.) and Physiology (P.T., S.V.-M, M.I.), Biocenter Oulu, University of Oulu, Oulu, Finland; and the Heart Institute (I.Sz.), Faculty of Medicine, University of Pécs, Pécs, Hungary; and the First Department of Internal Medicine (G.F., M.T.), Semmelweis University, Budapest, Hungary.

Correspondence to Heikki Ruskoaho, MD, PhD, Dept of Pharmacology and Toxicology, Faculty of Medicine, University of Oulu, PO Box 5000, 90014 University of Oulu, Finland. E-mail heikki.ruskoaho{at}oulu.fi

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
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.

Key Words: apelin • contractility • signal transduction • gene expression

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
G protein-coupled receptors (GPCRs) represent the largest group of transmembrane proteins responsible for transduction of a diverse array of extracellular signals. Recent progress in genome research unveiled a total of 300 GPCRs.¹ Of these, 160 GPCRs are activated by 97 known transmitters including biogenic amines, amino acids, peptides, nucleotides, fatty acid derivatives, and polypeptides. However, approximately 140 of these novel GPCRs do not bind any known endogenous ligand and are described as "orphan" GPCRs.¹

Recently, a ligand for one of the earliest orphan GPCRs has been revealed. APJ receptor, originally identified by O’Dowd et al² in 1993, shares the closest identity to the angiotensin II type 1 (AT₁) receptor ranging from 40% to 50% in the hydrophobic transmembrane regions, but does not bind angiotensin II. The putative endogenous ligand for the APJ receptor, apelin-36, was isolated from bovine stomach extracts by measuring extracellular acidification in a Chinese hamster ovary cell line expressing the human APJ receptor.³ Moreover, shorter synthetic C-terminal peptides consisting of 13 to 19 amino acids were found to exhibit significantly higher activity than apelin-36.^3,4

Although apelin and APJ mRNA have been found to be ubiquitously expressed in peripheral tissues as well as various regions of the central nervous system,^4–7 the exact function of apelin has not yet been established. Interestingly, sequence analysis of the mature apelin peptide revealed identity, albeit limited, to angiotensin II.⁵ In addition, APJ and AT₁ as well as apelin and angiotensinogen showed significant similarity in tissue distribution,⁵ suggesting that apelin and angiotensin II may affect the same biological processes. Indeed, intraperitoneal administration of apelin resulted in short-term increases in drinking behavior in rats,⁵ in parallel with the thirst-promoting effect of angiotensin II. In contrast to the well-established vasopressor effect of angiotensin II, intravenous injection of apelin lowered blood pressure in anesthetized rats.^5,8 Based on these preliminary results, one can anticipate that apelin, like angiotensin II, may have an important role in the regulation of cardiovascular homeostasis.

In the peripheral rat tissues, high levels of apelin mRNA^4–6 and moderate levels of APJ mRNA^6,7 were detected in the heart. Furthermore, quantitative autoradiography revealed the presence of specific APJ binding sites in human and rat myocardium with a comparable receptor density to AT₁.⁹ However, to date, there is no information available regarding the functional significance of the APJ-apelin system in the myocardium. Therefore, the objective of the present study was to characterize the direct cardiac effects of apelin as well as the underlying signaling pathways in vitro by using isolated perfused rat heart preparation and perforated patch-clamp recordings. Moreover, to test the potential pathophysiological importance of apelin, we studied the gene expression of apelin and APJ in vitro in cultured neonatal rat ventricular myocytes (NRVMs) subjected to mechanical stretch and in vivo in two models of chronic ventricular pressure overload.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Isolated Perfused Rat Heart Preparation
All protocols were reviewed and approved by the Animal Use and Care Committee of the University of Oulu. Male 7-week-old Sprague-Dawley (SD) rats (n=230) from the Center for Experimental Animals at the University of Oulu were used. Rats were decapitated and hearts were quickly removed and arranged for retrograde perfusion by the Langendorff technique as described previously.^10,11 The hearts were perfused with a modified Krebs-Henseleit bicarbonate buffer, pH 7.40, equilibrated with 95% O₂/5% CO₂ at 37°C. Hearts were perfused at a constant flow rate of 5.5 mL/min with a peristaltic pump (Minipuls 3, model 312). Heart rate was maintained constant (304±1 bpm) by atrial pacing using a Grass stimulator (model S88, 11 V, 0.5 ms). Contractile force (apicobasal displacement) was obtained by connecting a force displacement transducer (Grass Instruments, FT03) to the apex of the heart at an initial preload stretch of 2 g. In another set of experiments, left ventricular contractility was assessed by measuring isovolumic left ventricular pressure. Details of the methods and the experimental protocols are provided in an expanded Materials and Methods section, which can be found in the online data supplement available at http://www.circresaha.org.

Perforated Patch-Clamp Recordings
The whole-cell membrane currents were recorded from enzymatically isolated single adult ventricular myocytes by the Amphotericin B-perforated patch-clamp method¹² (for details see the online data supplement).

Mechanical Stretch of Neonatal Rat Ventricular Myocytes
NRVMs were subjected to cyclic stretch by means of the Flexercell computer-driven vacuum system¹³ (for details see the online data supplement).

Isolation of Cytoplasmic RNA and Northern Blot Analysis
Total RNA isolation and atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), and 18S RNA Northern blot analysis were performed as previously described.¹⁴

Real-Time Quantitative RT-PCR Analysis
Rat apelin, APJ receptor, and 18S RNA levels were measured by real-time quantitative RT-PCR analysis using Taqman chemistry on an ABI 7700 Genetic Analyzer (Applied Biosystems) as described previously.¹⁵ The sequences of the forward (F) and reverse (R) primers and probes (P) for RNA detection were as follows: apelin (F) 5'-CAAGGATCCCTTTGGCCC-3', (R) 5'-AGGAGAAGCTGGG-TCTCCAAG-3', (P) 5'-Fam-TCTTCCTGGCCACTCCTTGGACTGC-Tamra-3'; APJ (F) 5'-CTGCTGAGCATCATCGTGGT-3', (R) 5'-AGGGCCAGTGCAGCAAATT-3', (P) 5'-Fam-TGACCTTTGCCCT-GTGCTGGATGC-Tamra-3'; 18S (F) 5'-TGGTTGCAAAGCTGA-AACTTAAAG-3', (R) 5'-AGTCAAATTAAGCCGCAGGC-3', (P) 5'-Vic-CCTGGTGGTGCCCTTCCGTCA-Tamra-3'.

Statistical Analysis
Results are presented as mean±SEM. Data were analyzed with repeated measures- or 1-way ANOVA followed by Bonferroni post hoc test. Differences were considered statistically significant at the level of P<0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effect of Apelin on Contractility in Isolated Rat Hearts
Administration of apelin-16 (0.01 to 10 nmol/L) induced a dose-dependent increase in developed tension in the isolated rat heart preparation (Figure 1A; see also online Table 1, which can be found in the online data supplement available at http://www.circresaha.org). Maximal response to apelin was observed at the concentration of 1 nmol/L and the half-maximal effective concentration (EC₅₀) of apelin was 33.1±1.5 pmol/L. As shown in Figure 1B, the elevation of developed tension in response to apelin was gradual (F=72.1, P<0.001, apelin versus vehicle; for drug and time interaction, repeated measures ANOVA). A significant increase in contractility was observed 2 minutes after the start of apelin infusion, and the maximal increase was seen at approximately 24 minutes. The time-course of the effect of apelin was similar to those of the potent inotropic agents endothelin-1^11,16,17 and adrenomedullin ^10,11; however, it was markedly different from the rapidly developing, but short-lived effect of the ß-adrenergic receptor agonist isoproterenol (Figure 1B). The maximal responses to apelin, endothelin-1, and adrenomedullin were equal at the concentration of 1 nmol/L. Furthermore, the increase in developed tension induced by apelin was 69% of the maximal response to isoproterenol (10 µmol/L) (Figure 1B, online Table 1).

View larger version (14K): [in this window] [in a new window]   Figure 1. A, Effect of apelin (0.01 to 10 nmol/L) on developed tension (DT) in isolated perfused, paced rat hearts. B, Time course of the inotropic effect of apelin (1 nmol/L), endothelin-1 (1 nmol/L), adrenomedullin (1 nmol/L), and isoproterenol (10 µmol/L). After a control period, vehicle or the peptides were infused for 30 minutes and isoproterenol for 6 minutes. C, Effect of apelin on contractility at different levels of preload in isolated isovolumic rat hearts (for details see the online data supplement). Baseline dP/dtmax was 1273±52 mm Hg/s with no significant differences between the groups. Results are expressed as a percent change versus baseline values. Data are mean±SEM (n=5 to 7). *P<0.05 vs vehicle.

The apelin-induced increase in developed tension was not associated with significant changes in time-to-peak tension (before and after 1 nmol/L apelin: 57.0±2.6 versus 56.3±2.4 ms; P=NS) or time to 50% relaxation (40.9±1.0 versus 39.8±0.6 ms; P=NS). The resting tension (2.0±0.01 g) of the perfused hearts was not significantly affected by apelin (0.01 to 1 nmol/L), except that it induced a slight increase (2.2±0.03 g; P<0.05) at the highest concentration (10 nmol/L). Overall, changes in perfusion pressure induced by apelin (0.03 to 10 nmol/L) were small: eg, 1 nmol/L apelin slightly decreased the perfusion pressure from 32.4±1.6 to 30.6±1.4 mm Hg (P<0.001).

Effect of Preload on Apelin-Induced Positive Inotropic Effect
Next, we tested the effect of apelin on contractility at different levels of preload in isolated isovolumic rat hearts. The maximal derivative of isovolumic left ventricular pressure (dP/dt_max) was similar in the presence and absence of apelin (1 nmol/L) at a left ventricular end-diastolic pressure (LVEDP) of 1 or 5 mm Hg. However, when LVEDP was increased to 10 or 15 mm Hg in the presence of apelin, dP/dt_max was increased by 33% (P<0.05) and 35% (P<0.05) versus respective control values (Figure 1C). In contrast to the enhanced contractility, the diastolic function was not affected by apelin even at the highest level of LVEDP. The minimal derivative of isovolumic pressure (dP/dt_min: -832±40 versus -806±32 mm Hg/s; P=NS), time-from-peak systolic pressure to 60% relaxation (48.6±5.8 versus 50.3±2.4 ms; P=NS) or 90% relaxation (64.2±6.1 versus 66.5±3.1 ms; P=NS) and the time constant of exponential pressure decay (: 50.7±3.2 versus 51.2±2.7 ms; P=NS) did not differ significantly between vehicle and apelin-infused hearts at 15 mm Hg of LVEDP.

Specificity of the Apelin-Induced Positive Inotropic Effect
Because the APJ receptor shares sequence homology with AT₁ receptor,² we tested if the effect of apelin was mediated via angiotensin receptors. Infusion of CV-11974 (10 nmol/L), an AT₁ receptor antagonist,¹⁴ had no influence on developed tension (P=NS) and did not alter the positive inotropic response to 1 nmol/L apelin (P=NS; Figure 2A, online Table 2). To further characterize the specificity of the effect of apelin, we infused the peptide in the presence or absence of bosentan (1 µmol/L), an ET_A/ET_B endothelin receptor antagonist,¹¹ propranolol (1 µmol/L), a ß-adrenergic receptor blocker¹⁸ and prazosin (0.1 µmol/L), an -adrenergic receptor blocker.¹⁸ As shown in Figure 2, the apelin-induced increase in developed tension was not attenuated by the receptor antagonists (P=NS; online Table 2). None of the various antagonists affected baseline contractility (P=NS, Figure 2, online Table 2). Recently, apelin has been reported to lower blood pressure via a nitric oxide-dependent mechanism.⁸ Because low concentrations of nitric oxide can increase cardiac contractility,¹⁹ we tested the effect of inhibition of myocardial nitric oxide synthase on apelin-induced positive inotropic response. Infusion of L-NAME (300 µmol/L)¹¹ did not alter the apelin-induced increase in developed tension (43.3±2.1% versus 46.3±1.9%, apelin plus L-NAME versus apelin, n=4; P=NS).

View larger version (24K): [in this window] [in a new window]   Figure 2. Effects of (A) CV-11974 (10 nmol/L) and bosentan (1 µmol/L) and (B) prazosin (0.1 µmol/L) and propranolol (1 µmol/L) on apelin-induced (1 nmol/L) developed tension (DT) elevation. After a control period, vehicle or drugs were infused for 30 minutes. Results are expressed as a percent change vs baseline values. Data are mean±SEM (n=4 to 6).

Phospholipase C, Protein Kinase C, and Apelin-Induced Positive Inotropic Effect
An important mechanism for the regulation of cellular processes in cardiac myocytes involves phospholipase Cß-induced phosphoinositide hydrolysis with subsequent activation of protein kinase C.²⁰ To assess the involvement of phospholipase C (PLC) in the positive inotropic effect of apelin, we used U-73122, a potent inhibitor of this enzyme.²¹ Infusion of U-73122 (100 nmol/L) alone had no effect on contractile force (P=NS, Figure 3A, online Table 3). When U-73122 was infused in combination with apelin (1 nmol/L), it significantly decreased the apelin-induced inotropic effect throughout the entire experimental period, the maximal reduction being 68% at the end of 30 minutes of infusion time (F=18.6, P<0.001; Figure 3A, online Table 3). To examine whether activation of protein kinase C (PKC) contributes to the positive inotropic action of apelin, we evaluated the effects of the broad spectrum protein kinase inhibitor staurosporine,¹⁰ along with the specific PKC inhibitor GF-109203X.²² When apelin (1 nmol/L) was infused in the presence of staurosporine (10 nmol/L) or GF-109203X (90 nmol/L), the inotropic response was attenuated maximally by 77% (F=27.9; P<0.001) and 70% (F=28.1; P<0.01; Figure 3A, online Table 3), respectively. Infusion of staurosporine or GF-109203X alone had no influence on developed tension (P=NS, Figure 3A, online Table 3).

View larger version (26K): [in this window] [in a new window]   Figure 3. Effects of (A) U-73122 (100 nmol/L), staurosporine (10 nmol/L), and GF-109203X (90 nmol/L) and (B) zoniporide (1 µmol/L) and KB-R7943 (250 nmol/L) on apelin-induced (1 nmol/L) developed tension (DT) elevation. After a control period, vehicle or drugs were infused for 30 minutes. Results are expressed as a percent change versus baseline values. Data are mean±SEM (n=4 to 8).

Na⁺-H⁺ Exchange, Na⁺-Ca²⁺ Exchange, and Apelin-Induced Positive Inotropic Effect
Activation of PKC leads to phosphorylation of various cellular proteins including the sarcolemmal Na⁺-H⁺ exchanger.²³ To assess the contribution of Na⁺-H⁺ exchange (NHE) to the effect of apelin, we used MIA, an inhibitor of NHE.²⁴ Infusion of MIA (1 µmol/L) alone had no effect on contractile force (P=NS; online Table 3). When given together with apelin (1 nmol/L), MIA significantly attenuated the overall apelin-induced positive inotropic effect, the maximal reduction being 55% (F=12.7; P<0.001; online Table 3). Moreover, in the presence of zoniporide (1 µmol/L), a potent and selective inhibitor of NHE isoform-1 (NHE-1),²⁵ the inotropic response to apelin (1 nmol/L) was suppressed maximally by 60% (F=12.9, P<0.001; Figure 3B, online Table 3). Infusion of zoniporide alone had no influence on developed tension (P=NS, Figure 3B, online Table 3).

Accumulation of intracellular Na⁺ due to the activation of NHE may indirectly lead to an increase in intracellular Ca²⁺ level via sarcolemmal Na⁺-Ca²⁺ exchanger (NCX) working in reverse mode (Na⁺ out, Ca²⁺ in).²⁶ The role of NCX in apelin-induced positive inotropic effect was studied by using KB-R7943, a selective inhibitor of the reverse-mode NCX.^27,28 Administration of KB-R7943 (250 nmol/L) alone had no effect on baseline contractile force (P=NS; online Table 3). In contrast, KB-R7943 significantly attenuated the response to apelin (1 nmol/L) by 60% (F=18.5, P<0.001; Figure 3B, online Table 3). Next, we examined the effect of simultaneous inhibition of NHE and NCX on the inotropic effect of apelin. When given together with apelin (1 nmol/L), zoniporide (1 µmol/L) and KB-R7943 (250 nmol/L) significantly reduced the overall apelin-induced positive inotropic effect, the maximal reduction being 58% (F=13.8; P<0.001; Figure 3B, online Table 3). Infusion of zoniporide in combination with KB-R7943 had no effect on developed tension (P=NS; Figure 3B, online Table 3).

Effect of Apelin on Ca²⁺ and K⁺ Currents
We studied the effect of apelin on voltage-activated Ca²⁺ and K⁺ currents by performing amphotericin B-perforated patch-clamp recordings in isolated adult rat ventricular myocytes. Figure 4 shows the current-voltage relations established for the L-type Ca²⁺ current (I_Ca), the transient outward (I_to), and the sustained K⁺ current (I_K,sus) from the holding potential of -60 mV. These recordings showed that apelin (10 nmol/L) did not modulate I_Ca, I_to, or I_K,sus.

View larger version (16K): [in this window] [in a new window]   Figure 4. Current-voltage relationship of ICa (A) in the absence and presence of apelin (10 nmol/L). Current was elicited by test pulses stepping from -40 mV to +40 mV for 200 ms in 20-mV increments from the holding potential of -60 mV. Original current tracings before and after superfusion with apelin (from -60 mV to 0 mV) are shown in the inset. Current-voltage relationship for Ito (B) and IK,sus (C) in the absence and presence apelin (10 nmol/L). Current was elicited by test pulses stepping from -40 mV to +80 mV for 400 ms in 10-mV increments from the holding potential of -60 mV. Inset shows original current records (from -60 mV to +80 mV) before and after superfusion with apelin. Data are mean±SEM (n=4).

Effect of Mechanical Stretch on Apelin Gene Expression
We next studied the effect of mechanical load on apelin and APJ gene expression in vitro and in vivo. TaqMan RT-PCR analysis of cultured ventricular cells demonstrated that both apelin and APJ were mainly expressed in myocytes, whereas they were barely detectable in nonmyocyte-enriched cultures constituted mainly by ventricular fibroblasts. The expression of apelin and APJ in myocytes and nonmyocytes were 3.73±0.43 versus 0.27±0.13 (P<0.05, arbitrary units normalized to 18S RNA) and 4.64±0.65 versus 0.02±0.01 (P<0.05), respectively. Cyclic mechanical stretch of NRVMs in vitro for 12 or 24 hours decreased apelin and APJ mRNA levels by more than 50% (P<0.01; Figure 5A) and 30% (P<0.01), respectively. In contrast, BNP gene expression, used as a positive control for mechanical stretch,¹³ increased in a time-dependent manner (Figure 5B).

View larger version (29K): [in this window] [in a new window]   Figure 5. Effect of mechanical stretch on apelin (A) and BNP (B) gene expression in cultured NRVMs. Left ventricular levels of apelin (C) and ANP mRNA (D) in SD, dTG, WKY, and SHR animals. mRNA values are expressed as the ratio of apelin, BNP, or ANP mRNA to 18S as determined by quantitative reverse transcription-PCR analysis or Northern blot analysis, respectively. Data are mean±SEM (n=6 to 7). *P<0.05, P<0.01, and P<0.001 vs control by unpaired Student’s t test.

We also measured apelin and APJ gene expression in two models of chronic ventricular pressure overload. Double transgenic (dTG) rats harboring both the human angiotensinogen and human renin genes²⁹ and spontaneously hypertensive rats (SHR) displayed increased left ventricular weight to body weight index versus age-matched controls (4.11±0.15 versus 2.76±0.08 mg/g, P<0.001, dTG rats versus non-transgenic SD rats and 2.92±0.10 versus 1.66±0.10 mg/g, P<0.001, SHR versus Wistar-Kyoto [WKY] rats). Left ventricular levels of apelin mRNA were 33% lower in dTG rats (P<0.05, dTG rats versus non-transgenic SD rats; Figure 5C) and 62% in SHR (P<0.001, SHR versus WKY rats; Figure 5C), respectively, whereas APJ mRNA levels remained unchanged (data not shown). In contrast, ANP gene expression, a hallmark of hypertrophy, was 9.9- and 4.2-fold higher in dTG rats and SHR versus their respective controls (P<0.001; Figure 5D).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study provides the first evidence for the functional importance of apelin, a putative ligand for the APJ orphan receptor in the heart. Among the peripheral rat tissues, high levels of apelin mRNA^4–6 and considerable levels of APJ mRNA^6,7 have been found in the myocardium. Our results show that both apelin and APJ are mainly expressed in cardiac myocytes, indicating the existence of a potential autocrine/paracrine regulatory loop in the myocardium. Indeed, our results demonstrate that apelin exerts a potent, dose-dependent positive inotropic effect in vitro, in the isolated perfused rat heart preparation. On a molar basis, endothelin-1¹⁶ and adrenomedullin¹⁰ have been considered previously to be the most potent stimulators of cardiac contractility with EC₅₀ values of 50 pmol/L. Because apelin was active in the subnanomolar range, with an EC₅₀ value of 33 pmol/L, apelin appears to be among the most potent endogenous positive inotropic substances yet identified. Because plasma concentrations of apelin are approximately 23 pmol/L in rats⁴ and local levels may be even higher because of active synthesis, circulating or locally produced apelin may play an important role in the regulation of cardiac function. Notably, the apelin-induced increase in developed tension was 69% of the maximal inotropic response to isoproterenol and it was comparable to the effect of endothelin-1 and adrenomedullin in isolated perfused rat hearts. Of particular interest was our finding that apelin possessed a slowly developing but sustained inotropic response that clearly differed from the classical ß-adrenergic effect, which develops rapidly, usually over a matter of seconds, suggesting that apelin may modulate the inotropic responsiveness of the heart over a different time frame (eg, from minutes to hours).

APJ receptor shares modest sequence homology with AT₁ receptor.² However, it is unlikely that the effect of apelin is mediated via angiotensin receptors, because CV-11947, a specific AT₁ receptor antagonist, failed to attenuate the inotropic response to apelin. Moreover, the effect of apelin remained unchanged in the presence of an ET_A/ET_B endothelin receptor antagonist, - and ß-adrenergic receptor blockers, and a nitric oxide synthase inhibitor. Thus, it appears that apelin can bind and activate its own receptors in the heart specifically, independent of release of endogenous angiotensin II, endothelin, catecholamines or nitric oxide.

Prolonged activation of PKC by diacylglycerol, a product of PLCß-induced phosphoinositide hydrolysis, has been considered to be an important pathway in cellular responses in cardiac myocytes.²⁰ Our results suggest that activation of PLC and PKC are involved in the positive inotropic effect of apelin, because the apelin-induced increase in developed tension was markedly attenuated by U-73122, a PLC inhibitor, and staurosporine and GF-109203X, a nonselective and a specific inhibitor of PKC, respectively. Activated PKC can phosphorylate a wide spectrum of cellular proteins including the sarcolemmal NHE.²³ Previously, the apelin-induced promotion of extracellular acidification rate in Chinese hamster ovary cells expressing the APJ receptor was suppressed by MIA, a nonspecific inhibitor of NHE.⁷ In our experiments, MIA and zoniporide, a highly selective inhibitor of NHE-1, significantly attenuated the inotropic response to apelin, suggesting that activation of NHE, at least in part, contributes to the effect of apelin. Stimulation of the NHE can lead to intracellular alkalinization and sensitization of cardiac myofilaments to intracellular Ca²⁺. On the other hand, NHE-mediated accumulation of intracellular Na⁺ can indirectly promote a rise in intracellular levels of Ca²⁺ via reverse mode NCX.²⁶ In the present study, KB-R7943, a selective inhibitor of the reverse mode NCX markedly reduced the apelin-induced increase in developed tension. Based on the concentration of KB-R7943 (250 nmol/L), it is likely that the compound acted selectively, because it inhibits the voltage-gated Na⁺ current, Ca²⁺ current, and the inward rectifier K⁺ current with IC₅₀ vales of 14, 8, and 7 µmol/L,³⁰ respectively, and has no effect on NHE up to a concentration of 30 µmol/L.²⁷ Our observation that simultaneous administration of zoniporide and KB-R7943 could not attenuate further the inotropic response to apelin may suggest that NHE and NCX are proximal and distal components, respectively, of a contiguous signaling pathway. It is noteworthy that both mechanisms have been shown previously to play an important role in the positive inotropic effect of endothelin-1^17,28 and angiotensin II.³¹ Activation of protein kinase A leads to a robust increase in I_Ca, which plays a critical role in the positive inotropic response to ß-adrenergic stimulation. In contrast, the effect of stimulation of PKC on L-type Ca²⁺ channels is controversial. I_Ca has been reported to be either modestly increased, decreased, or unchanged by ET-1, angiotensin II, and ₁-adrenergic agonists.³² In perforated patch-clamp experiments, we did not find any evidence for modulation of I_Ca by apelin. In addition, apelin did not alter voltage-activated K⁺ currents (I_to and I_K,sus). These results suggest that activation of NHE and NCX contributes to the inotropic effect of apelin, whereas voltage-activated Ca²⁺ and K⁺ currents are not involved. The finding that 40% of the apelin-induced positive inotropic effect remained unaffected even after combined inhibition of NHE and NCX indicates the existence of additional signaling mechanisms. Apelin may also affect the properties of more downstream elements of the contractile machinery such as the Ca²⁺ affinity of troponin C or the actomyosin crossbridge cycling rate.³³

Previously, it has been reported that the APJ receptor expressed in Chinese hamster ovary cells is coupled to pertussis toxin-sensitive G proteins (G_i or G_o protein).^7,34 Our preliminary results showed that pertussis toxin pretreatment (25 µg/kg IP, 48 hours before the experiment)²² partly reduced the apelin-induced positive inotropic response (26.8±4.4% versus 46.3±1.9%, apelin with and without pertussis toxin pretreatment, n=4; P<0.05). Because the PLC-PKC pathway is coupled to pertussis toxin-insensitive G proteins (G_q/11),³⁵ the signaling mechanisms activated by apelin may involve both pertussis toxin-insensitive and -sensitive G proteins.

Taking into account the mechanism of action, one may speculate a potential pathophysiological relevance of apelin. An abrupt increase in hemodynamic load triggers a series of adaptive mechanisms in the myocardium.³⁶ Stretch of cardiac muscle generates a biphasic force response: an initial change that occurs almost immediately and a second slowly developing phase. The first phase has been attributed to increased myofilament Ca²⁺ responsiveness, whereas stretch-induced activation of NHE and NCX are likely to mediate the slow-force response.^26,37,38 It is intriguing that the time-course and the signaling pathways underlying the effect of apelin show similarities to the slow-force response. When stretch persists for longer periods, genes are switched on and off that eventually lead to cardiac hypertrophy and failure.³⁶ Notably, apelin gene expression was markedly downregulated in cultured ventricular myocytes subjected to mechanical stretch and in vivo in 2 models of chronic ventricular pressure overload. Thus, as a feedback mechanism, the cardiac effects of apelin may be offset by its decreased synthesis, and in the long run, it can contribute to the deterioration of cardiac function. Further studies are required to test the hypothesis that restoration of myocardial apelin synthesis can rescue the failing heart.

In summary, this is the first report showing that apelin exerts a potent positive inotropic effect in vitro. Our results suggest that the inotropic response to apelin may involve activation of PLC, PKC, sarcolemmal NHE, and NCX.

	Acknowledgments

 
This work was supported by the Academy of Finland, the Sigrid Juselius Foundation, the Finnish Foundation for Cardiovascular Research, the Hungarian Research Foundation (OTKA: F035213), and the Ministry of Health of Hungary (ETT: 304/2000 and 264/2001).

Received August 29, 2001; revision received August 1, 2002; accepted August 2, 2002.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Civelli O, Nothacker H, Saito Y, Wang Z, Lin SH, Reinscheid RK. Novel neurotransmitters as natural ligands of orphan G-protein-coupled receptors. Trends Neurosci. 2001; 24: 230–237.[CrossRef][Medline] [Order article via Infotrieve]

2. O’Dowd BF, Heiber M, Chan A, Heng HH, Tsui LC, Kennedy JL, Shi X, Petronis A, George SR, Nguyen T. A human gene that shows identity with the gene encoding the angiotensin receptor is located on chromosome 11. Gene. 1993; 136: 355–360.[CrossRef][Medline] [Order article via Infotrieve]

3. Tatemoto K, Hosoya M, Habata Y, Fujii R, Kakegawa T, Zou MX, Kawamata Y, Fukusumi S, Hinuma S, Kitada C, Kurokawa T, Onda H, Fujino M. Isolation and characterization of a novel endogenous peptide ligand for the human APJ receptor. Biochem Biophys Res Commun. 1998; 251: 471–476.[CrossRef][Medline] [Order article via Infotrieve]

4. Kawamata Y, Habata Y, Fukusumi S, Hosoya M, Fujii R, Hinuma S, Nishizawa N, Kitada C, Onda H, Nishimura O, Fujino M. Molecular properties of apelin: tissue distribution and receptor binding. Biochim Biophys Acta. 2001; 1538: 162–171.[Medline] [Order article via Infotrieve]

5. Lee DK, Cheng R, Nguyen T, Fan T, Kariyawasam AP, Liu Y, Osmond DH, George SR, O’Dowd BF. Characterization of apelin, the ligand for the APJ receptor. J Neurochem. 2000; 74: 34–41.[CrossRef][Medline] [Order article via Infotrieve]

6. O’Carroll AM, Selby TL, Palkovits M, Lolait SJ. Distribution of mRNA encoding B78/apj, the rat homologue of the human APJ receptor, and its endogenous ligand apelin in brain and peripheral tissues. Biochim Biophys Acta. 2000; 1492: 72–80.[Medline] [Order article via Infotrieve]

7. Hosoya M, Kawamata Y, Fukusumi S, Fujii R, Habata Y, Hinuma S, Kitada C, Honda S, Kurokawa T, Onda H, Nishimura O, Fujino M. Molecular and functional characteristics of APJ: tissue distribution of mRNA and interaction with the endogenous ligand apelin. J Biol Chem. 2000; 275: 21061–21067.[Abstract/Free Full Text]

8. Tatemoto K, Takayama K, Zou MX, Kumaki I, Zhang W, Kumano K, Fujimiya M. The novel peptide apelin lowers blood pressure via a nitric oxide-dependent mechanism. Regul Pept. 2001; 99: 87–92.[CrossRef][Medline] [Order article via Infotrieve]

9. Katugampola SD, Maguire JJ, Matthewson SR, Davenport AP. [¹²⁵I]-(Pyr¹)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. Br J Pharmacol. 2001; 132: 1255–1260.[CrossRef][Medline] [Order article via Infotrieve]

10. Szokodi I, Kinnunen P, Tavi P, Weckström M, Tóth M, Ruskoaho H. Evidence for cAMP-independent mechanisms mediating the effects of adrenomedullin, a new inotropic peptide. Circulation. 1998; 97: 1062–1070.[Abstract/Free Full Text]

11. Kinnunen P, Szokodi I, Nicholls MG, Ruskoaho H. Impact of NO on ET-1- and AM-induced inotropic responses: potentiation by combined administration. Am J Physiol Regul Integr Comp Physiol. 2000; 279: R569–R575.[Abstract/Free Full Text]

12. Rae J, Gates P, Watsky M. Low access resistance perforated patch recording using amphotericin B. J Neurosci Methods. 1991; 37: 15–26.[CrossRef][Medline] [Order article via Infotrieve]

13. Liang F, Gardner DG. Mechanical strain activates BNP gene transcription through a p38/NF-B-dependent mechanism. J Clin Invest. 1999; 104: 1603–1612.[Medline] [Order article via Infotrieve]

14. Magga J, Vuolteenaho O, Marttila M, Ruskoaho H. Endothelin-1 is involved in stretch-induced early activation of B-type natriuretic peptide gene expression in atrial but not in ventricular myocytes: acute effects of mixed ET_A/ET_B and AT₁ receptor antagonists in vivo and in vitro. Circulation. 1997; 96: 3053–3062.[Abstract/Free Full Text]

15. Majalahti-Palviainen T, Hirvinen M, Tervonen V, Ilves M, Ruskoaho H, Vuolteenaho O. Gene structure of a new cardiac peptide hormone: a model for heart-specific gene expression. Endocrinology. 2000; 141: 731–740.[Abstract/Free Full Text]

16. Kelly RA, Eid H, Krämer BK, O’Neill M, Liang BT, Reers M, Smith TW. Endothelin enhances the responsiveness of adult rat ventricular myocytes to calcium by a pertussis toxin-sensitive pathway. J Clin Invest. 1990; 86: 1164–1171.[Medline] [Order article via Infotrieve]

17. Krämer BK, Smith TW, Kelly RA. Endothelin and increased contractility in adult rat ventricular myocytes: role of intracellular alkalosis induced by activation of the protein kinase C-dependent Na⁺-H⁺ exchanger. Circ Res. 1991; 68: 269–279.[Abstract/Free Full Text]

18. Talosi L, Kranias EG. Effects of -adrenergic receptor stimulation on activation of protein kinase C and phosphorylation of proteins in intact rabbit hearts. Circ Res. 1992; 70: 670–678.[Abstract/Free Full Text]

19. Kojda G, Kottenberg K, Nix P, Schlüter KD, Piper HM, Noack E. Low increase in cGMP induced by organic nitrates and nitrovasodilators improves contractile response of rat ventricular myocytes. Circ Res. 1996; 78: 91–101.[Abstract/Free Full Text]

20. Nishizuka Y. Protein kinase C and lipid signaling for sustained cellular responses. FASEB J. 1995; 9: 484–496.[Abstract]

21. Fulton D, McGiff JC, Quilley J. Role of phospholipase C and phospholipase A2 in the nitric oxide-induced vasodilator effect of bradykinin in the rat perfused heart. J Pharmacol Exp Ther. 1996; 278: 518–526.[Abstract/Free Full Text]

22. Hu K, Nattel S. Mechanisms of ischemic preconditioning in rat hearts: involvement of _1B-adrenoceptors, pertussis toxin-sensitive G proteins, and protein kinase C. Circulation. 1995; 92: 2259–2265.[Abstract/Free Full Text]

23. Karmazyn M, Gan XT, Humphreys RA, Yoshida H, Kusumoto K. The myocardial Na⁺-H⁺ exchange: structure, regulation, and its role in heart disease. Circ Res. 1999; 85: 777–786.[Abstract/Free Full Text]

24. Moffat MP, Karmazyn M. Protective effects of the potent Na/H exchange inhibitor methylisobutyl amiloride against post-ischemic contractile dysfunction in rat and guinea-pig hearts. J Mol Cell Cardiol. 1993; 25: 959–971.[CrossRef][Medline] [Order article via Infotrieve]

25. Knight DR, Smith AH, Flynn DM, MacAndrew JT, Ellery SS, Kong JX, Marala RB, Wester RT, Guzman-Perez A, Hill RJ, Magee WP, Tracey WR. A novel sodium-hydrogen exchanger isoform-1 inhibitor, zoniporide, reduces ischemic myocardial injury in vitro and in vivo. J Pharmacol Exp Ther. 2001; 297: 254–259.[Abstract/Free Full Text]

26. Kentish JC. A role for the sarcolemmal Na⁺-H⁺ exchanger in the slow force response to myocardial stretch. Circ Res. 1999; 85: 658–660.[Free Full Text]

27. Iwamoto T, Watano T, Shigekawa M. A novel isothiourea derivative selectively inhibits the reverse mode of Na⁺-Ca²⁺ exchange in cells expressing NCX1. J Biol Chem. 1996; 271: 22391–22397.[Abstract/Free Full Text]

28. Yang HT, Sakurai K, Sugawara H, Watanabe T, Norota I, Endoh M. Role of Na⁺-Ca²⁺ exchange in endothelin-1-induced increases in Ca²⁺ transient and contractility in rabbit ventricular myocytes: pharmacological analysis with KB-R7943. Br J Pharmacol. 1999; 126: 1785–1795.[CrossRef][Medline] [Order article via Infotrieve]

29. Bohlender J, Menard J, Luft FC, Ganten D. Dose effects of human renin in rats transgenic for human angiotensinogen. Hypertension. 1997; 29: 1031–1038.[Abstract/Free Full Text]

30. Watano T, Kimura J, Morita T, Nakanishi H. A novel antagonist, No. 7943, of the Na⁺-Ca²⁺ exchange current in guinea-pig cardiac ventricular cells. Br J Pharmacol. 1996; 119: 555–563.[Medline] [Order article via Infotrieve]

31. Fujita S, Endoh M. Influence of a Na⁺-H⁺ exchange inhibitor ethylisopropylamiloride, a Na⁺-Ca²⁺ exchange inhibitor KB-R7943 and their combination on the increases in contractility and Ca²⁺ transient induced by angiotensin II in isolated adult rabbit ventricular myocytes. Naunyn Schmiedebergs Arch Pharmacol. 1999; 360: 575–584.[CrossRef][Medline] [Order article via Infotrieve]

32. Kamp TJ, Hell JW. Regulation of cardiac L-type calcium channels by protein kinase A and protein kinase C. Circ Res. 2000; 87: 1095–1102.[Abstract/Free Full Text]

33. Endoh M. Regulation of myocardial contractility by a downstream mechanism. Circ Res. 1998; 83: 230–232.[Free Full Text]

34. Masri B, Lahlou H, Mazarguil H, Knibiehler B, Audigier Y. Apelin (65–77) activates extracellular signal-regulated kinases via a PTX-sensitive G protein. Biochem Biophys Res Commun. 2002; 290: 539–545.[CrossRef][Medline] [Order article via Infotrieve]

35. Neer EJ. Heterotrimeric G proteins: organizers of transmembrane signals. Cell. 1995; 80: 249–257.[CrossRef][Medline] [Order article via Infotrieve]

36. Tavi P, Laine M, Weckström M, Ruskoaho H. Cardiac mechanotransduction: from sensing to disease and treatment. Trends Pharmacol Sci. 2001; 22: 254–260.[CrossRef][Medline] [Order article via Infotrieve]

37. Alvarez BV, Pérez NG, Ennis IL, Camilión de Hurtado MC, Cingolani HE. Mechanisms underlying the increase in force and Ca²⁺ transient that follow stretch of cardiac muscle: a possible explanation of the Anrep effect. Circ Res. 1999; 85: 716–722.[Abstract/Free Full Text]

38. Pérez NG, de Hurtado MC, Cingolani HE. Reverse mode of the Na⁺-Ca²⁺ exchange after myocardial stretch: underlying mechanism of the slow force response. Circ Res. 2001; 88: 376–382.[Abstract/Free Full Text]

This article has been cited by other articles:

				B. C. Frier, D. B. Williams, and D. C. Wright The effects of apelin treatment on skeletal muscle mitochondrial content Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2009; 297(6): R1761 - R1768. [Abstract] [Full Text] [PDF]

				D. N. Charo, M. Ho, G. Fajardo, M. Kawana, R. K. Kundu, A. Y. Sheikh, T. P. Finsterbach, N. J. Leeper, K. V. Ernst, M. M. Chen, et al. Endogenous regulation of cardiovascular function by apelin-APJ Am J Physiol Heart Circ Physiol, November 1, 2009; 297(5): H1904 - H1913. [Abstract] [Full Text] [PDF]

				D. E Henley, F. Buchanan, R. Gibson, J. A Douthwaite, S. A Wood, W. W Woltersdorf, J. R Catterall, and S. L Lightman Plasma apelin levels in obstructive sleep apnea and the effect of continuous positive airway pressure therapy J. Endocrinol., October 1, 2009; 203(1): 181 - 188. [Abstract] [Full Text] [PDF]

				E. Calderon-Sanchez, C. Delgado, G. Ruiz-Hurtado, A. Dominguez-Rodriguez, V. Cachofeiro, M. Rodriguez-Moyano, A. M. Gomez, A. Ordonez, and T. Smani Urocortin induces positive inotropic effect in rat heart Cardiovasc Res, September 1, 2009; 83(4): 717 - 725. [Abstract] [Full Text] [PDF]

				J. J. Maguire, M. J. Kleinz, S. L. Pitkin, and A. P. Davenport [Pyr1]Apelin-13 Identified as the Predominant Apelin Isoform in the Human Heart: Vasoactive Mechanisms and Inotropic Action in Disease Hypertension, September 1, 2009; 54(3): 598 - 604. [Abstract] [Full Text] [PDF]

				M. L. Kirby Why Don't They Beat?: Cripto, Apelin/APJ, and Myocardial Differentiation Circ. Res., July 31, 2009; 105(3): 211 - 213. [Full Text] [PDF]

				R. A.P. Weir, K. S. Chong, J. R. Dalzell, C. J. Petrie, C. A. Murphy, T. Steedman, P. B. Mark, T. A. McDonagh, H. J. Dargie, and J. J.V. McMurray Plasma apelin concentration is depressed following acute myocardial infarction in man Eur J Heart Fail, June 1, 2009; 11(6): 551 - 558. [Abstract] [Full Text] [PDF]

				I. Falcao-Pires, N. Goncalves, T. Henriques-Coelho, D. Moreira-Goncalves, R. Roncon-Albuquerque Jr., and A. F. Leite-Moreira Apelin decreases myocardial injury and improves right ventricular function in monocrotaline-induced pulmonary hypertension Am J Physiol Heart Circ Physiol, June 1, 2009; 296(6): H2007 - H2014. [Abstract] [Full Text] [PDF]

				O. Kunduzova, N. Alet, N. Delesque-Touchard, L. Millet, I. Castan-Laurell, C. Muller, C. Dray, P. Schaeffer, J. P. Herault, P. Savi, et al. Apelin/APJ signaling system: a potential link between adipose tissue and endothelial angiogenic processes FASEB J, December 1, 2008; 22(12): 4146 - 4153. [Abstract] [Full Text] [PDF]

				B. Rentzsch, M. Todiras, R. Iliescu, E. Popova, L. A. Campos, M. L. Oliveira, O. C. Baltatu, R. A. Santos, and M. Bader Transgenic Angiotensin-Converting Enzyme 2 Overexpression in Vessels of SHRSP Rats Reduces Blood Pressure and Improves Endothelial Function Hypertension, November 1, 2008; 52(5): 967 - 973. [Abstract] [Full Text] [PDF]

				I. Szokodi, R. Kerkela, A.-M. Kubin, B. Sarman, S. Pikkarainen, A. Konyi, I. G. Horvath, L. Papp, M. Toth, R. Skoumal, et al. Functionally Opposing Roles of Extracellular Signal-Regulated Kinase 1/2 and p38 Mitogen-Activated Protein Kinase in the Regulation of Cardiac Contractility Circulation, October 14, 2008; 118(16): 1651 - 1658. [Abstract] [Full Text] [PDF]

				A. Kasai, N. Shintani, H. Kato, S. Matsuda, F. Gomi, R. Haba, H. Hashimoto, M. Kakuda, Y. Tano, and A. Baba Retardation of Retinal Vascular Development in Apelin-Deficient Mice Arterioscler Thromb Vasc Biol, October 1, 2008; 28(10): 1717 - 1722. [Abstract] [Full Text] [PDF]

				B. Chandrasekaran, O. Dar, and T. McDonagh The role of apelin in cardiovascular function and heart failure Eur J Heart Fail, August 1, 2008; 10(8): 725 - 732. [Abstract] [Full Text] [PDF]

				M. Karmazyn, D. M. Purdham, V. Rajapurohitam, and A. Zeidan Signalling mechanisms underlying the metabolic and other effects of adipokines on the heart Cardiovasc Res, July 15, 2008; 79(2): 279 - 286. [Abstract] [Full Text] [PDF]

				C. Wang, J.-F. Du, F. Wu, and H.-C. Wang Apelin decreases the SR Ca2+ content but enhances the amplitude of [Ca2+]i transient and contractions during twitches in isolated rat cardiac myocytes Am J Physiol Heart Circ Physiol, June 1, 2008; 294(6): H2540 - H2546. [Abstract] [Full Text] [PDF]

				M. Lorenz, N. Hellige, P. Rieder, H.-T. Kinkel, C. Trimpert, A. Staudt, S. B. Felix, G. Baumann, K. Stangl, and V. Stangl Positive inotropic effects of epigallocatechin-3-gallate (EGCG) involve activation of Na+/H+ and Na+/Ca2+ exchangers Eur J Heart Fail, May 1, 2008; 10(5): 439 - 445. [Abstract] [Full Text] [PDF]

				M. Azizi, X. Iturrioz, A. Blanchard, S. Peyrard, N. De Mota, N. Chartrel, H. Vaudry, P. Corvol, and C. Llorens-Cortes Reciprocal Regulation of Plasma Apelin and Vasopressin by Osmotic Stimuli J. Am. Soc. Nephrol., May 1, 2008; 19(5): 1015 - 1024. [Full Text] [PDF]

				A. Y. Sheikh, H. J. Chun, A. J. Glassford, R. K. Kundu, I. Kutschka, D. Ardigo, S. L. Hendry, R. A. Wagner, M. M. Chen, Z. A. Ali, et al. In vivo genetic profiling and cellular localization of apelin reveals a hypoxia-sensitive, endothelial-centered pathway activated in ischemic heart failure Am J Physiol Heart Circ Physiol, January 1, 2008; 294(1): H88 - H98. [Abstract] [Full Text] [PDF]

				A. J. Glassford, P. Yue, A. Y. Sheikh, H. J. Chun, S. Zarafshar, D. A. Chan, G. M. Reaven, T. Quertermous, and P. S. Tsao HIF-1 regulates hypoxia- and insulin-induced expression of apelin in adipocytes Am J Physiol Endocrinol Metab, December 1, 2007; 293(6): E1590 - E1596. [Abstract] [Full Text] [PDF]

				K. Kuba, L. Zhang, Y. Imai, S. Arab, M. Chen, Y. Maekawa, M. Leschnik, A. Leibbrandt, M. Markovic, J. Schwaighofer, et al. Impaired Heart Contractility in Apelin Gene Deficient Mice Associated With Aging and Pressure Overload Circ. Res., August 17, 2007; 101(4): e32 - e42. [Abstract] [Full Text] [PDF]

				K. Higuchi, T. Masaki, K. Gotoh, S. Chiba, I. Katsuragi, K. Tanaka, T. Kakuma, and H. Yoshimatsu Apelin, an APJ Receptor Ligand, Regulates Body Adiposity and Favors the Messenger Ribonucleic Acid Expression of Uncoupling Proteins in Mice Endocrinology, June 1, 2007; 148(6): 2690 - 2697. [Abstract] [Full Text] [PDF]

				V.-P. Ronkainen, J. J. Ronkainen, S. L. Hanninen, H. Leskinen, J. L. Ruas, T. Pereira, L. Poellinger, O. Vuolteenaho, and P. Tavi Hypoxia inducible factor regulates the cardiac expression and secretion of apelin FASEB J, June 1, 2007; 21(8): 1821 - 1830. [Abstract] [Full Text] [PDF]

				J.-C. Zhong, X.-Y. Yu, Y. Huang, L.-M. Yung, C.-W. Lau, and S.-G. Lin Apelin modulates aortic vascular tone via endothelial nitric oxide synthase phosphorylation pathway in diabetic mice Cardiovasc Res, June 1, 2007; 74(3): 388 - 395. [Abstract] [Full Text] [PDF]

				P. Francia, A. Salvati, C. Balla, P. De Paolis, E. Pagannone, M. Borro, G. Gentile, M. Simmaco, L. De Biase, and M. Volpe Cardiac resynchronization therapy increases plasma levels of the endogenous inotrope apelin Eur J Heart Fail, March 1, 2007; 9(3): 306 - 309. [Abstract] [Full Text] [PDF]

				B. Gurzu, B. Cristian Petrescu, M. Costuleanu, and G. Petrescu Interactions between apelin and angiotensin II on rat portal vein Journal of Renin-Angiotensin-Aldosterone System, December 1, 2006; 7(4): 212 - 216. [Abstract] [PDF]

				R. R. van Kimmenade, J. L. Januzzi Jr, P. T. Ellinor, U. C. Sharma, J. A. Bakker, A. F. Low, A. Martinez, H. J. Crijns, C. A. MacRae, P. P. Menheere, et al. Utility of Amino-Terminal Pro-Brain Natriuretic Peptide, Galectin-3, and Apelin for the Evaluation of Patients With Acute Heart Failure J. Am. Coll. Cardiol., September 19, 2006; 48(6): 1217 - 1224. [Abstract] [Full Text] [PDF]

				B. Masri, N. Morin, L. Pedebernade, B. Knibiehler, and Y. Audigier The Apelin Receptor Is Coupled to Gi1 or Gi2 Protein and Is Differentially Desensitized by Apelin Fragments J. Biol. Chem., July 7, 2006; 281(27): 18317 - 18326. [Abstract] [Full Text] [PDF]

				C. J Charles, M. T Rademaker, and A M. Richards Apelin-13 induces a biphasic haemodynamic response and hormonal activation in normal conscious sheep. J. Endocrinol., June 1, 2006; 189(3): 701 - 710. [Abstract] [Full Text] [PDF]

				K. S. Chong, R. S. Gardner, J. J. Morton, E. A. Ashley, and T. A. McDonagh Plasma concentrations of the novel peptide apelin are decreased in patients with chronic heart failure Eur J Heart Fail, June 1, 2006; 8(4): 355 - 360. [Abstract] [Full Text] [PDF]

				T. Hashimoto, M. Kihara, J. Ishida, N. Imai, S.-i. Yoshida, Y. Toya, A. Fukamizu, H. Kitamura, and S. Umemura Apelin Stimulates Myosin Light Chain Phosphorylation in Vascular Smooth Muscle Cells Arterioscler Thromb Vasc Biol, June 1, 2006; 26(6): 1267 - 1272. [Abstract] [Full Text] [PDF]

				A.-M. O'Carroll, S. J Lolait, and G. M Howell Transcriptional regulation of the rat apelin receptor gene: promoter cloning and identification of an Sp1 site necessary for promoter activity J. Mol. Endocrinol., February 1, 2006; 36(1): 221 - 235. [Abstract] [Full Text] [PDF]

				P. T. Ellinor, A. F. Low, and C. A. MacRae Reduced apelin levels in lone atrial fibrillation Eur. Heart J., January 2, 2006; 27(2): 222 - 226. [Abstract] [Full Text] [PDF]

				S. Crowley, S. Gurley, M. Oliverio, A. Pazmino, R Griffiths, P. Flannery, R. Spurney, H-S Kim, O Smithies, T. Le, et al. Is the Kidney Always the Cause of Hypertension?: Distinct Roles for the Kidney and Systemic Tissues in Blood Pressure Regulation by the Renin-Angiotensin System. J Clin Invest 115: 1092-1099, 2005 J. Am. Soc. Nephrol., June 1, 2005; 16(6): 1525 - 1532. [Full Text] [PDF]

				J. P. Goetze and R. Videbaek More hormones spilt in heart failure: linking renal sympathetic activation to clinical outcome Eur. Heart J., May 1, 2005; 26(9): 861 - 862. [Full Text] [PDF]

				J. Boucher, B. Masri, D. Daviaud, S. Gesta, C. Guigne, A. Mazzucotelli, I. Castan-Laurell, I. Tack, B. Knibiehler, C. Carpene, et al. Apelin, a Newly Identified Adipokine Up-Regulated by Insulin and Obesity Endocrinology, April 1, 2005; 146(4): 1764 - 1771. [Abstract] [Full Text] [PDF]

				J.-C. Zhong, D.-Y. Huang, G.-F. Liu, H.-Y. Jin, Y.-M. Yang, Y.-F. Li, X.-H. Song, and K. Du Effects of all-trans retinoic acid on orphan receptor APJ signaling in spontaneously hypertensive rats Cardiovasc Res, February 15, 2005; 65(3): 743 - 750. [Abstract] [Full Text] [PDF]

				D. K. Lee, V. R. Saldivia, T. Nguyen, R. Cheng, S. R. George, and B. F. O'Dowd Modification of the Terminal Residue of Apelin-13 Antagonizes Its Hypotensive Action Endocrinology, January 1, 2005; 146(1): 231 - 236. [Abstract] [Full Text] [PDF]

				G. A. Losano On the cardiovascular activity of apelin Cardiovasc Res, January 1, 2005; 65(1): 8 - 9. [Full Text] [PDF]

				E. A. Ashley, J. Powers, M. Chen, R. Kundu, T. Finsterbach, A. Caffarelli, A. Deng, J. Eichhorn, R. Mahajan, R. Agrawal, et al. The endogenous peptide apelin potently improves cardiac contractility and reduces cardiac loading in vivo Cardiovasc Res, January 1, 2005; 65(1): 73 - 82. [Abstract] [Full Text] [PDF]

				C. J. Pemberton, H. Tokola, Z. Bagi, A. Koller, J. Pontinen, A. Ola, O. Vuolteenaho, I. Szokodi, and H. Ruskoaho Ghrelin induces vasoconstriction in the rat coronary vasculature without altering cardiac peptide secretion Am J Physiol Heart Circ Physiol, October 1, 2004; 287(4): H1522 - H1529. [Abstract] [Full Text] [PDF]

				M. F. Berry, T. J. Pirolli, V. Jayasankar, J. Burdick, K. J. Morine, T. J. Gardner, and Y. J. Woo Apelin Has In Vivo Inotropic Effects on Normal and Failing Hearts Circulation, September 14, 2004; 110(11_suppl_1): II-187 - II-193. [Abstract] [Full Text] [PDF]

				J. Ishida, T. Hashimoto, Y. Hashimoto, S. Nishiwaki, T. Iguchi, S. Harada, T. Sugaya, H. Matsuzaki, R. Yamamoto, N. Shiota, et al. Regulatory Roles for APJ, a Seven-transmembrane Receptor Related to Angiotensin-type 1 Receptor in Blood Pressure in Vivo J. Biol. Chem., June 18, 2004; 279(25): 26274 - 26279. [Abstract] [Full Text] [PDF]

				O. Tenhunen, B. Sarman, R. Kerkela, I. Szokodi, L. Papp, M. Toth, and H. Ruskoaho Mitogen-activated Protein Kinases p38 and ERK 1/2 Mediate the Wall Stress-induced Activation of GATA-4 Binding in Adult Heart J. Biol. Chem., June 4, 2004; 279(23): 24852 - 24860. [Abstract] [Full Text] [PDF]

				D. K. Lee, A. J. Lanca, R. Cheng, T. Nguyen, X. D. Ji, F. Gobeil Jr., S. Chemtob, Susan. R. George, and B. F. O'Dowd Agonist-independent Nuclear Localization of the Apelin, Angiotensin AT1, and Bradykinin B2 Receptors J. Biol. Chem., February 27, 2004; 279(9): 7901 - 7908. [Abstract] [Full Text] [PDF]

				M. M. Chen, E. A. Ashley, D. X.F. Deng, A. Tsalenko, A. Deng, R. Tabibiazar, A. Ben-Dor, B. Fenster, E. Yang, J. Y. King, et al. Novel Role for the Potent Endogenous Inotrope Apelin in Human Cardiac Dysfunction Circulation, September 23, 2003; 108(12): 1432 - 1439. [Abstract] [Full Text] [PDF]

				J. Piuhola, M. Makinen, I. Szokodi, and H. Ruskoaho Dual role of endothelin-1 via ETA and ETB receptors in regulation of cardiac contractile function in mice Am J Physiol Heart Circ Physiol, June 5, 2003; 285(1): H112 - H118. [Abstract] [Full Text] [PDF]

				M. Avkiran and R. S Haworth Regulatory effects of G protein-coupled receptors on cardiac sarcolemmal Na+/H+ exchanger activity: signalling and significance Cardiovasc Res, March 15, 2003; 57(4): 942 - 952. [Abstract] [Full Text] [PDF]

				J. Piuhola, I. Szokodi, P. Kinnunen, M. Ilves, R. deChatel, O. Vuolteenaho, and H. Ruskoaho Endothelin-1 Contributes to the Frank-Starling Response in Hypertrophic Rat Hearts Hypertension, January 1, 2003; 41(1): 93 - 98. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 91/5/434    most recent 01.RES.0000033522.37861.69v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Szokodi, I. Articles by Ruskoaho, H. Search for Related Content PubMed PubMed Citation Articles by Szokodi, I. Articles by Ruskoaho, H. Related Collections Contractile function Receptor pharmacology