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 (ETR A and ETR B) in atrial tissues, with a concomitant increase in ANP secretion. The ETR A or ETR B 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.
Hypoxia is a common pathological condition in most cardiac diseases such as myocardial hypertrophy and infarction [
Endothelin (ET)-1 is the predominant ET isopeptide in the heart [
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.
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 [
The acute hypoxic atrial model was prepared by replacing O2 gas with N2 gas and normal HEPES buffer with N2 saturated HEPES buffer. The criteria for successful hypoxic atrial modeling included markedly increased secretion of ANP and significant inhibitory atrial dynamics.
To investigated effect of hypoxia on atrial ANP secretion, ET-1 release and ETRs levels, 25 of rabbits were randomly divided in to atrial ANP secretion and dynamics ((a) and (b), n = 5), ET-1 release ((c), n = 5), ETRA ((d), n = 5), ETRB ((e), n = 5) and control groups ((c), (d) and (e), n = 5) (
For roles of ET receptors antagonists on hypoxia-induced atrial ANP secretion experiments, 24 of rabbits were randomly divided in to hypoxia ((Aa), (Ba) and (Ca), n = 6), BQ123 ((Aa), n = 6), BQ788 ((Ba), n = 6) and combined treatment of BQ123 plus BQ788 groups ((Ca), n = 6) (
Another experiments for roles of ERK and Akt antagonists on hypoxia-induced atrial ANP secretion, 12 of rabbits were randomly divided in to hypoxia ((Aa) and (Ba), n = 6), PD98059 ((Aa), n = 6), LY294002 groups ((Ba), n = 6) (
Finally, to determined roles of ET receptors antagonists on hypoxia-induced atrial ERK and Akt levels, 25 of rabbits were randomly divided in to control ((a) and (b) = 5), hypoxia ((a) and (b), n = 5), BQ123 ((a) and (b), n = 5), BQ788 ((a) and (b), n = 5) and combined treatment of BQ123 plus BQ788 groups ((a) and (b), n = 5) (
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.
A separate series of experiments was performed to investigate the mechanism by which endogenous ET-1 regulates acute hypoxia-induced ANP secretion. After the control period, the treatment cycle was followed by four cycles of infusion of the treatment agent plus hypoxia. The treatment agents were as follows: 1) the ETRA antagonist BQ123 (0.3 μmol/L; n = 6); 2) the ETRB antagonist BQ788 (0.3 μmol/L; n = 6); 3) the MAPK/ERK inhibitor PD098059 (30 μmol/L; n = 6); 4) the PI3K/Akt inhibitor LY294002 (30 μmol/L; n = 6).
The levels of ET-1 and immunoreactive ANP in the perfusate were measured by a specific radioimmunoassay as described previously [
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
concentrations were determined using the Bradford Protein Assay Kit. Solubilized protein was denatured in Lane Maker Loading buffer, assayed by 10% or 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride filter membranes (Beyotime institute of Biotechnology, China). Membranes were blocked with 5% nonfat dry milk in phosphate buffer solution (PBST) at room temperature. After 2 hours, the membrane were incubated with goat polyclonal anti-ETA (1:100; Santa Cruz Biotechnology, Hong Kong), rabbit polyclonal anti-ETB (1:1000; Abcam, Hong Kong), rabbit polyclonal p-Akt (1:1000; Abcam Hong Kong) or p-ERK (1:1000; Cell Signaling Technology, Shanghai, China), all with rabbit polyclonal β-actin (1:1000; ComWin Biotech, Beijing, China) at 4˚C, overnight. Membranes were incubated with the secondary antibodies (1:2000) for 2 h at room temperature. After extensive washing with PBST, bands were visualized with the ECL Plus Western blotting detection system (ECL Western Blot Kit ComWin Biotech, Beijing, China) and then quantified using Image J software.
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 significantly (P < 0.001 vs. control; n = 6) increased and decreased ANP secretion and atrial stroke volume, respectively in isolated perfused beating rabbit atria (
Acute hypoxia markedly (P < 0.001 vs. control; n = 6) promoted the release of atrial ET-1 (
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 mmol/L; for ETRA) and BQ788 (0.3 mmol/L; for ETRB) significantly (P < 0.001 vs. hypoxia, n = 6) attenuated hypoxia-mediated secretion of ANP (
The MAPK/ERK and PI3K/Akt signaling pathways are considered important players in the effects mediated by ET-1 [
To confirm that ERK and Akt were involved in ET-1-mediated regulation of hypoxia-induced ANP secretion, we investigated the effects of the ETR antagonists on the expression of p-ERK and p-Akt. Acute hypoxia markedly (P < 0.001 vs. control; n = 5) increased atrial p-ERK levels (
p-Akt levels were markedly (P < 0.001 vs. control; n = 5) increased by hypoxia (
The present study showed that acute hypoxia significantly promoted the release of atrial ET-1 and upregulated the expression of ETRA and ETRB. 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 [
Accumulating evidence has suggested that MAPK/ERK and PI3K/Akt are the most important downstream signaling pathways of ETRs [
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.
This work was supported by the National Natural Science Foundation of China (No. 81360061 and 81260035) and Jilin Provincial Research Foundation for Basic Research, China (No. 20130101133JC).
ChaochaoBian,DazhiDing,HonghuaJin,LipingLiu,LanHong,BairiCui,11,XunCui,11,11, (2016) Endogenous Endothelin-1 Regulates Hypoxia-Induced Atrial Natriuretic Peptide Secretion by Activating the MAPK/ERK and PI3K/Akt Signaling Pathways in Isolated Beating Rabbit Atria. Journal of Biosciences and Medicines,04,45-53. doi: 10.4236/jbm.2016.41006