Endogenous Endothelin-1 Regulates Hypoxia-Induced Atrial Natriuretic Peptide Secretion by Activating the MAPK / ERK and PI 3 K / Akt Signaling Pathways in Isolated Beating Rabbit Atria

The present study investigated a possible mechanism for endogenous endothelin-1 (ET-1) regulation of atrial natriuretic peptide (ANP) secretion in isolated perfused acute hypoxic rabbit atria. Acute hypoxia significantly enhanced the release of ET-1 and the expression of the ET receptor (ETR) type A and B (ETRA and ETRB) in atrial tissues, with a concomitant increase in ANP secretion. The ETRA or ETRB antagonist, BQ123 (0.3 μmol/L) or BQ788 (0.3 μmol/L), respectively attenuated hypoxia-induced ANP secretion. Both antagonists significantly attenuated the levels of hypoxiainduced atrial phosphorylated (p)-extracellular signal-regulated kinase (ERK) and p-protein kinase B (Akt). The ERK and Akt inhibitors, PD098059 (30 μmol/L) and LY294002 (30 μmol/L), respectively mimicked the effect of the ETR antagonists. These results demonstrated that acute hypoxia-mediated atrial ET-1 regulated ANP secretion through ETR and the subsequent mitogenactivated protein kinase (MAPK)/ERK and ETR-phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathways. These pathways may mediate atrial endocrine functions under hypoxic conditions. These authors contributed equally to this work. Co-corresponding author. Corresponding author.


Introduction
Hypoxia is a common pathological condition in most cardiac diseases such as myocardial hypertrophy and infarction [1] [2].Numerous studies have clearly shown that hypoxia stimulates the secretion of atrial natriuretic peptide (ANP), resulting in cellular adaptation to hypoxia and protection of the ischemic heart [3]- [5].However, the mechanism by which hypoxia induces ANP secretion is not well known.
Endothelin (ET)-1 is the predominant ET isopeptide in the heart [6].ET-1 is an important regulator of cardiac function, and its cardiac and plasma levels are elevated in myocardial infarction, hypertension, and heart failure [7]- [9].ET-1 has been shown to the most potent stimulus for ANP secretion [10].We have previously reported that ET-1 significantly promotes ANP secretion via the endothelin receptor type B (ETRB) mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathway under normoxic conditions in isolated perfused beating rabbit atria [11].Moreover, other studies have demonstrated that hypoxia significantly increases the production of ET-1 [2] [12] and that hypoxia-induced ET-1 is involved in the regulation of ANP secretion [10] [13].However, results for the role of ETRs, ETRA, and ETRB, in the regulation of hypoxia-induced ANP secretion are conflicting.Skvorak et al. [14] have shown that a period of anoxia in the isolated perfused rat atria causes a dramatic increase in ANP secretion, which is almost entirely blocked by the ETRA antagonist BQ123.However, Zhang et al. [13] have reported that the nonselective ETR antagonist bosentan, but not BQ123, significantly attenuates hypoxia-induced ANP release.Overall, the mechanism by which endogenous ET-1 regulates hypoxia-induced ANP secretion remains to be defined.
The purpose of the present study was to investigate the mechanism by which hypoxia-induced endogenous ET-1 regulates ANP secretion in isolated perfused beating rabbit atria.We investigated the role of ETR and their downstream signaling pathways, MAPK/ERK and phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt), on the regulation of hypoxia-mediated ANP secretion.

Isolated Perfused Beating Rabbit Atrial Model Preparation
The atria (mean wet weight of 167.00 ± 4.2 mg) of adult New Zealand white rabbits of either sex were used.The isolated perfused beating left atrial model was prepared as previously described [15].The beating atrium was perfused with HEPES buffer solution ([in mmol/L]: 118 NaCl, 4.7 KCl, 2.5 CaCl 2 , 1.2 MgCl 2 , 25 NaHCO 3 ,10 glucose, 10 HEPES, 0.1% bovine serum albumin; pH 7.4).The peristaltic pump (1 mL/min) allowed atrial pacing for measurements of the changes in atrial volume (stroke volume) and the secretion of ET-1 and ANP.Transmural electrical field stimulation with a luminal electrode was operated at 1.5 Hz (0.3 ms, 30 -40 V).The perfused atrium was supplied with sufficient oxygen during the entire process.

Acute Hypoxic Atrial Model Preparation
The acute hypoxic atrial model was prepared by replacing O 2 gas with N 2 gas and normal HEPES buffer with N 2 saturated HEPES buffer.The criteria for successful hypoxic atrial modeling included markedly increased secretion of ANP and significant inhibitory atrial dynamics.

Experimental Protocols
Each atrium was perfused for 60 min to stabilize the parameters of ET-1, ANP secretion, and atrial dynamics.The control cycle (12 min as an experimental cycle) was followed by the infusion of hypoxic buffer for four cycles.The changes in atrial dynamics were recorded by the RM-6240 system.ET-1 and ANP levels were determined by radioimmunoassay in the perfusate, which was collected at 2 min intervals (at 4˚C).Immediately after perfusion, the atrial tissues were collected, frozen in liquid nitrogen, and stored at −80˚C for future use in western immunoblotting.

Radioimmunoassay of ANP Concentrations
The levels of ET-1 and immunoreactive ANP in the perfusate were measured by a specific radioimmunoassay as described previously [16].The amounts of secreted ET-1 and ANP were expressed as ng/min/g of wet atrial tissue.

Western Immunoblot Analysis
Protein from left atrial tissues was tested by western blotting.The atrial tissues were homogenated in radio immunoprecipitation assay lysis buffer (Solarbio institute of Biotechnology, Shanghai, China), and protein

Statistical Analysis
All data are presented as mean ± SEM and were analyzed by the one-way analysis of variance followed by the Dunnett's multiple comparison test.An unpaired t-test was also applied.Statistical significance was defined as P < 0.05.

Acute Hypoxia Potently Promoted Atrial ANP, ET-1 Secretion and the Expression of ETRs
Acute hypoxia significantly (P < 0.001 vs. control; n = 6) increased and decreased ANP secretion and atrial stroke volume, respectively in isolated perfused beating rabbit atria (Figure 1(a) and Figure 1(b)).These results suggested that the acute hypoxia model in the isolated perfused beating rabbit atria was successful.Acute hypoxia markedly (P < 0.001 vs. control; n = 6) promoted the release of atrial ET-1 (Figure 1(c)).Furthermore, western blotting analysis revealed that the expression of ETR A and ETR B was markedly (P < 0.01 and P < 0.001, respectively vs. control; n = 6) increased in perfused beating rabbit atrial tissues under acute hypoxic conditions (Figure 1(d) and Figure 1(e)).These results indicated that acute hypoxia promoted the release of atrial ET-1 and upregulated the expression of both ETR types.

Effect of ETRs on Hypoxia-Induced ANP Secretion
We then used ETR antagonists to determine if ET-1-mediated stimulation of ETRs is involved in the regulation of hypoxia-induced secretion of ANP.BQ123 (0.3 µmol/L; for ETR A ) and BQ788 (0.3 µmol/L; for ETR B ) significantly (P < 0.001 vs. hypoxia, n = 6) attenuated hypoxia-mediated secretion of ANP (Figure 2(Ab) and Figure 2(Bb)).The inhibitory effect was markedly (P < 0.001 vs. hypoxia; n = 6) enhanced with the co-treatment of both antagonists (Figure 2(Cb)).These results indicated that endogenous ET-1-mediated stimulation of ETRs was involved in the regulation of hypoxia-induced secretion of ANP.

Effects of the ERK and Akt Signaling Pathways on Regulation of Hypoxia-Induced Secretion of ANP
The MAPK/ERK and PI3K/Akt signaling pathways are considered important players in the effects mediated by ET-1 [16]- [18].Therefore, we investigated the effects of ERK and Akt on hypoxia-induced secretion of ANP.The data showed that both inhibitors of ERK and Akt, PD98059 (30.0 µmol/L) and LY294002 (30.0 µmol/L), significantly attenuated hypoxia-induced secretion of ANP (P < 0.001 vs. hypoxia; n = 6. Figure 3(Ab) and Figure 3(Bb)), and both inhibitors mimicked the effects of BQ123 and BQ788 on the regulation of hypoxiainduced ANP secretion.These results indicated that the MAPK/ERK and PI3K/Akt signaling pathways may have been involved in endogenous ET-1-mediated regulation of hypoxia-induced ANP secretion.

Discussion
The present study showed that acute hypoxia significantly promoted the release of atrial ET-1 and upregulated the expression of ETR A and ETR B .We also demonstrated that hypoxia-mediated production of ET-1 caused the activation of ETRs resulting in the regulation of ANP secretion via the MAPK/ERK and -PI3K/Akt signaling pathways.
Previous studies have demonstrated that hypoxia is the predominant stimulus for the production of ET-1 in endothelial cells and the primary cause for the increased synthesis and release of ET-1 during myocardial ischemia [19]- [22].Increased ET-1 secretion during myocardial ischemia has been linked to enhanced contractility in the failing heart [23] and to heart failure [24].Recently, the hypoxia-inducible factor-1α (HIF-1α) binding site in the pre-ET-1 gene in cardiomyocytes has been identified as a site of synthesis and action for ET-1 in hypoxia-induced expression of ET-1 [25]- [27].These findings were also reported in the present study, in which acute hypoxia significantly increased atrial ET-1 release in the isolated perfused beating rabbit atria.Furthermore, our findings revealed that acute hypoxia markedly up regulated the expression of ETR A and ETR B and potently stimulated the secretion of ANP, which was significantly attenuated by the ETR A or ETR B antagonist, BQ123 or ETR B BQ788, respectively.These results indicated that acute hypoxia-mediated endogenous ET-1 contributed to the hypoxia-induced secretion of ANP through the activation of ETR A and ETR B .In our previous study, both ETR A and ETR B were found to be involved in the regulation of ET-1-mediated ANP secretion in isolated perfused beating rabbit normoxic atria [11].Therefore, these findings suggest that both ETRs play an important role in the regulation of ET-1-mediated ANP secretion in either normoxic or hypoxic conditions.Moreover, Zhang et al. [13] have reported that the nonselective ETR antagonist bosentan, and not BQ123, significantly attenuates hypoxia-induced secretion of ANP.However, Skvorak et al. [14] have shown that anoxiaincreased secretion of ANP is almost entirely blocked by BQ123 in isolated perfused rat atria.These discrepancies may be attributed to species or model differences, or different levels of endogenous ET-1.
Accumulating evidence has suggested that MAPK/ERK and PI3K/Akt are the most important downstream signaling pathways of ETRs [16]- [18].Furthermore, findings from our previous study have revealed that the MAPK/ERK and PI3K/Akt signaling pathways are the most important regulators of ANP secretion in normoxic or hypoxic conditions [11].However, whether or not these two pathways are involved in the regulation of endogenous ET-1 on hypoxia-induced atrial ANP secretion is unclear.Therefore, in the present study, we investigated the effect of endogenous ET-1 on the hypoxia-induced expression of p-ERK and p-Akt in isolated rabbit atria.The results showed that both ETR antagonists significantly down regulated the hypoxia-mediated increased expression of both phosphorylated proteins, concomitantly with a marked attenuation in the secretion of ANP.The regulation of hypoxia-induced ANP secretion by the p-ERK and Akt antagonists (PD98059 and LY294002, respectively) was mimicked by the ETR antagonists.These findings corroborate with previous results suggesting that the MAPK/ERK and PI3K/Akt signaling pathways contributed to the regulation of endogenous ET-1 on hypoxia-induced ANP secretion [11].

Conclusion
In conclusion, our findings indicate that acute hypoxia significantly promotes the production of atrial ET-1 which, in turn, regulates ANP secretion through the activation of ETR and the subsequent MAPK/ERK and -PI3K/Akt signaling pathways.These two pathways may be the mechanisms responsible for atrial endocrine effects under hypoxic conditions.