Mechanisms Of Acute Hypoxia-induced ANP Secretion In Isolated Beating Atria

Hypoxia caused by the reduce of intracellular oxygen concentration or limited availability of oxygen, it is a common phenomenon of physiology and pathophysiology in the important tissues, including heart, kidney and brain. Myocardial ischemia, the most common cause of cardiac hypoxia in clinical medicine, occurs when oxygen delivery can not meet myocardial metabolic requirements in the heart and has been observed in patients of general clinical with coronary artery disease, myocardial hypertrophy, myocardial infarction and heart failure. In order to adapt to hypoxia environment, mammalian cells lead to management several hypoxia-inducible genes including vascular endothelial cell growth factor (VEGF), various glycolytic enzymes, glucose transporter1, hemeoxygenase-1(HO-1) and Endothelin-1(ET-1) and so on, which are regulated by hypoxia-inducible factor-1(HIF-1).HIF-1is a transcription factor regulated by intracellular O2tension. It is a heterodimer which composed of HIF-1a and HIF-1β subunit. According to most studies showed, HIF-1activity determined by a subunit. So far, the expression of approximately100genes directly or indirectly regulated by HIF-1and participate in essential physiological processes such as angiogenesis, vascular remodeling, vasoconstriction, glucose and energy metabolism, erythropoiesis, iron homeostasis, pH regulation, cell proliferation and survival. Then, HIF-I is found that play an important protective roles in acute, chronic, and intermittent hypoxia as well as in the ischemia-reperfusion injury.HEF-1α activity is modulated by protein kinase phosphorylation (PKP). Mitogen-activated protein kinases (MAPKs)/extracellular signal regulated kinase (ERK) and phosphoinositide3kinase (PI3K)/Akt (also been known as protein kinase B, PKB), the most important regulators of HIF-1α, play the key roles in phosphorylation of Ser/Thr and to protecte for heart damage by ischemia or hypoxia. ANP as a potent diuretic, natriuretic peptide hormone participates to against cardiac hypertrophy, fibrosis, proliferation and heart failure. It is synthesized and released primarily from atrial myocytes. Numerous studies showed that hypoxia is a essential stimuli of the atrial ANP secretion. However, there are controversial resultes in the regulation of HIF-1on hypoxia-induced ANP secretion. Some studies suggested that HIF-1is not a directly factor of ANP secretion, but another studies claimed that HIF-1directly activates the ANP gene promoter or indirectly promotes ANP secretion through VEGF in the early phases in hypoxia. In addition, the roles of MAPK/ERK and PI3K/Akt signaling pathway on the regulation of hypoxia-induced ANP secretion.The purpose of the present study, therefore, is to investigate the effect of acute hypoxia on the atrial ANP secretion and to determine the relationships between MAPK/ERK, PI3K/Akt signaling and hypoxia-induced ANP release in perfused beating rabbit atria.1. Acute hypoxia significantly increased atrial ANP secretion (P<0.001vs control), concomitantly with decreased atrial pulse pressure (P<0.001vs control) and atrial stroke volume (P<0.001vs control) in the perfused beating rabbit atria;2. Hypoxia increased the mRNA (P<0.01vs control) and protein level (P<0.001vs control) of HIF-1α in atrial tissue;3. The mRNA (P<0.01, P<0.001vs hypoxia, respectively) and protein level (P<0.01vs hypoxia, also) of HIF-1a were obviously down-regulated by rotenone (0.5μmol) or CAY10585(10μmo), inhibitors of HIF-1α. Hypoxia-promoted atrial ANP secretion was also significantly reduced (P<0.001vs hypoxia alone) in the presence inhibitors of HIF-1α without changed in hypoxia-inhibited atrial dynamics (P<0.001vs control);4. The mRNA (P<0.01, P<0.001vs hypoxia, respectively) and protein level (P<0.001vs hypoxia, also) of HIF-la were significantly down-regulated by PD98059(30μmol) and LY294002(30μmol), inhibitors of MAPK/ERK and PDK/Akt respectively, and hypoxia-promoted atrial ANP secretion was also significantly inhibited (P<0.001vs hypoxia alone) by inhibitors without changed in hypoxia-inhibited atrial dynamics (P<0.001vs control).These results indicated that: 1. Acute hypoxia significantly increased atrial ANP secretion through HIF-la in isolated beating rabbit atria;2. MAPK/ERK and PI3K/Akt signaling pathway regulated hypoxia-induced ANP release by controlling HIF-1α activity. Atrial natriuretic peptide (ANP) is mainly synthesized and secreted in the atrial myocytes. ANP as a cardiac hormone not only involved in the sodium diuresis, regulation of blood pressure and electrolyte balance, but also to protectes cardiac myocytes. A number of studies have demonstrated that hypoxia stimulates atrial ANP secretion and precipitates celles to adapt the hypoxic environment. However, the mechanism of hypoxia-induced ANP secretion is not clear.Protein tyrosine kinases (PTKs), the most important enzyme for the protein phosphorylation, may lead to phosphorylation of tyrosine residues in various substrates. So far, it is known that there are about90receptor tyrosine kinases (RTKs) genes and43RTKs-dependent genes in human genome, which activate some growth factors or cytokines and transduce the signaling into intracellular. PTKs have emerged as important roles of maintain steady state in normal cells and regulators of critical cellular processes, such as proliferation and differentiation, cell survial and metabolism, cell migration and cell apoptosis. There are two kind types of PTKs in celles that one of them is RTKs also been known as functional receptor and another one is non-receptor tyrosine kinases (NRTKs), free form.Numerous studies have discovered that mutations or aberrant in PTKs may lead to activation of their intracellular signaling and to results of many disorders. At present, it has been demonstrated that PTKs are involved in the regulation of inflammation, malignant cell transformation and angiogenesis of the several diseases, such as diabetes and cancer. However, effects of PTKs on the regulation of cardiovascular functions is not well known. Recent studies showed that hypoxia inducible factor-la (HIF-1α) regulated genes, including the platelet derived growth factor (PDGF), insulin-like growth factor-1(IGF-1), vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) activation were closely related with PTKs signal transduction.Mitogen-activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK) and Phosphoinositide-3kinase (PI3K)/Akt signaling have been recognized the important down stream pathways of PTKs.. It has been demonstrated that the MAPK/ERK and PI3K/Akt regulate hypoxia-induced ANP secretion by controlling the activity of HIF-1α. Simultaneously, the other studies showed that PTKs play the crucial roles in the regulation of HIF-la activity. Nevertheless, effects of PTKs on regulation of hypoxia-induced atrial ANP secretion is not known.In addition, cyclooxygenases (COX) is the must enzymes in the synthesis of prostaglandins (PGs). COX catalysis arachidonic acid (AA) into PGs and play potent autocrine or paracrine function in almost mammalian cardiac tissue. Some experiments confirmed that PTKs signaling pathway involved in the synthesis of PGs, in which the expression of COX has related with PTKs signaling, especially COX-2. Although there are controversial results in the relationships between PGs and ANP secretion but the common have believed that PGs is a important stimuli of ANP secretion. However, effects of PTKs-COX signaling on the regulation of hypoxia-induced ANP secretion is not well known.The purpose of the present study, therefore, is to investigate the effect of acute hypoxia on the atrial ANP secretion and the relationships between PTKs as well as its downstream signaling pathways and hypoxia induced ANP release in the perfused beating rat atria.The results of the present study were showed that:1. Acute hypoxia significantly increased ANP secretion (P<0.001vs control), concomitantly with decreased atrial pulse pressure (P<0.001vs control) in perfused beating rat atria; 2. Hypoxia-induced atrial ANP secretion was significantly decreased (P<0.001vs hypoxia alone) by genistein (3μmol), an inhibitor of PTKs, without changed hypoxia-decreased atrial pulse pressure (P<0.001vs control alone);3. Genistein potently inhibited hypoxia-increased expression of p-ERK (P<0.01vs hypoxia alone) and almost blocked the hypoxia-induced expression of p-Akt (P<0.OOlvs hypoxia alone);4. PD98059(30μmol/L), an inhibitor of MAPK/ERK, accentuated the inhibitory effect of genistein on the regulation of hypoxia-induced atrial ANP secretion (P<0.001vs genistein+hypoxia) and the LY294002(30μmol/L), an inhibitor of PI3K/Akt, similarly to the PD98059(P<0.001vs genistein+hypoxia); PD98059or LY294002failed to modulation of hypoxia-dcreased atrial PP (P<0.001vs control respectively);5. HIF-1α inhibitors rotenone (0.5μmol) or CAY10585(10μmol) also accentuated the inhibitory effect of genistein on the regulation of hypoxia-induced atrial ANP secretion (P<0.001vs genistein+hypoxia) without changed hypoxia-decreased atrial PP (P<0.001vs control respectively);6. Hypoxia increased expression of HIF-1α and GATA4(P<0.001vs control) and were obviously inhibited by genistein, or concomitantly with PD98059, LY294002as well as inhibitors HIF-1α(P<0.05vs hypoxia alone respectively);7. Indomethacin (10μmol/L), an inhibitor of COX, significantly inhibited the effect of hypoxia-increased atrial ANP secretion (P<0.001vs hypoxia alone) without changed hypoxia-decreased atrial PP (P<0.001vs control);8. Genistein, PD98059and LY294002were accentuated the inhibitory effect of indomethacin on regulation of hypoxia-increased atrial ANP secretion (all P<0.001vs hypoxia alone) and failed to modulation of hypoxia-decreased atrial PP (P<0.001vs control);9. Hypoxia markedly up regulated the expression of atrial COX-2and indomethacin and/or genistein were significantly inhibited hypoxia-induced atrial COX-2express. These results indicated that:1. HIF-la regulates acute hypoxia-increased ANP secretion via GATA4gene express in isolated beating rat atria;2. PTKs-MAPK/ERK or-PBK/Akt signaling pathways regulate hypoxia-induced atrial ANP release by controlling HIF-la activity;3. COX has been involved in the regulation of PTKs on hypoxia-induced ANP release.