The Effect Of Hypoxia On ANP Release And The Cardioprotective Action Of ANP

Roles of endothelin-1and JNK signaling pathway on hypoxia-induced ANP release in beating rabbit atriaSince the discovery of atrial natriuretic peptide (ANP), the heart is no longer considered only as an efficient pump but also as an important endocrine gland. The heart generates and secretes natriuretic peptides. The family of natriuretic peptides hormone is stored in human and mammals is characterized by a conserved ring structure of17amino acids sharing the same sequence.There are four family members in the natriuretic peptide family (NPs), including ANP, brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP) and dendroaspis natriuretic peptide (DNP). ANP is the first member of the NPs family. It is known that all of the NPs members have important biological effects, such as diuresis and natriuresis, regulating body fluid and blood pressure. As a cardiac hormone synthesized and stored primarily in the cardiac atria, ANP plays important roles in modulating blood pressure and cardiovascular homeostasis by combining with natriuretic peptide receptors which exist in cardiovascular system, central nervous system, lymph tissue, reproductive organs and kidney. Recently, numerous studies have focused on the cardioprotective effects of ANP, such as modulation of cardiac hypertrophy, fibrosis, and the development of hypoxic pulmonary hypertension.Many factors are closely related to the regulation of atrial ANP secretion. From a physiological standpoint, the most important factor of ANP secretion is mechanical stretching of the atria which is normally occurs when extracellular fluid volume or blood volume are elevated. In addition, several vasoconstrictors such as endothelin and angiotensin Ⅱ contribute to increase ANP secretion. A dramatic increase of ANP release is produced by cardiac ischemia and hypoxia has also been reported. Even several studies have indicated that hypoxia is related with atrial ANP secretion not only in vivo but also in vitro. Hypoxia has a direct stimulatory effect on ANP release from atrial cardiomyocytes in vitro and oxytocin rather than stretch itself may induce ANP release from rat cardiac myocytes. However, the mechanism by which hypoxia increases ANP release is not well known.Hypoxia can activate many signaling pathways hypoxia-inducible factor-1 (HIF-1) which has been known to present in almost cell types. It is tightly regulated by O2availability and regulates the expression of numerous genes, including vasoactive substances such as ET-1. It is well known that ET-1may strongly stimulate ANP secretion. Lew and Baertschi demonstrated this important relationship by co-culturing cardiac myocytes and endothelial cells, which resulted in enhanced ANP secretion. Fukuda et al showed that endothelin increases ANP secretion and up-regulates ANP messenger RNA in isolated rat cardiac myocytes. Skvorak et al demonstrated that much of the increase in ANP secretion induced by ischemia could be attributed to the release of endothelins. Therefore, it can be hypothesized that HIF-1a and/or its target gene ET-1may be involved in the regulation of hypoxia-induced atrial ANP secretion.Under hypoxic conditions, activities and gene expressions of the HIF-1a may be affected by cell O2concentrations as well as the mitogen-activated-protein-kinases (MAPKs) signaling pathway. There are at least three subfamilies of MAPKs: extracellular signal-regulated kinases (ERK1/2), Jun amino-terminal kinases (INK) and p38MAP kinases. Among them, JNK is well known for its roles in stress responses and oncogenesis. However, the relationship between MAPK and hypoxia-induced ANP release remains to be defined.Based on these evidences, the purpose of the present study is to confirm the effects of hypoxia on the atrial ANP secretion and the relationship between ET-1or MAPK-JNK and hypoxia induced ANP release. Experiments were performed in the perfused beating rabbit atrial model and the ANP as well as ET-1levels in the atrial perfusates were measured by radioimmnoassay. The results of the present study are as follows.1. Hypoxia significantly increased atrial ANP secretion (P<0.001vs control), but concomitantly decreased atrial pulse pressure (P<0.001vs control) and stroke volume (P<0.001vs control).2. Hypoxia-induced atrial ET secretion was also enhanced (P<0.05vs control). In the presence of BQ123and BQ788, the inhibitors of ETA-R (type A of endothelin receptor) and ETB-R (type B of endothelin receptor) respectively, hypoxia-induced increase in atrial ANP secretion was markedly attenuated (P<0.001vs hypoxia alone), concomitantly decreased atrial pulse pressure and stroke volume (P<0.001vs control).3. SP600125, an inhibitor of JNK, significantly inhibited the effect of hypoxia-increased atrial ANP secretion as well as ET release (P<0.001vs hypoxia alone), and the inhibitory effect of SP600425on hypoxia-increased ANP secretion was stronger than BQ123+BQ788olne (P<0.05vs BQ123+BQ788).4. The inhibitory effect of SP600125+BQ123+BQ788on the hypoxia-induced ANP secretion was similar to that of SP600125alone (P<0.001vs hypoxia alone; P>0.05vs SP600125alone).These results indicate that:1. Hypoxia significantly increased atrial ANP secretion in isolated beating rabbit atria;2. HIF-1target gene ET-1was involved in the regulation of hypoxia-increased ANP secretion;3. The MAPK-JNK signaling pathway plays an important role in hypoxia-mediated ANP release. Heart is no longer considered only as an efficient pump but also as an important endocrine organ, becausre the heart generates and secretes atrial natriuretic peptide (ANP). It is well known that ANP have important biological effects, such as diuresis and natriuresis, modulating blood pressure and cardiovascular homeostasis by combining with natriuretic peptide receptors. ANP binds to NPR-A receptor which is a member of the membrane-bound guanylyl cyclase family and exerts its biological effects by increasing cGMP levels.ANP has been reported to protect animal and human hearts through the cGMP/PKG signaling pathway when given at the time of reperfusion, suggesting that ANP can prevent myocardial reperfusion injury. However, the exact mechanism by which ANP prevents reperfusion injury remains to be defined.’Mitochondrial permeability transition pore (mPTP) opening has been demonstrated to play a critical role in reperfusion injury and prevention of the mPTP opening leads to cardioprotection against reperfusion injury. The activation of cGMP/PKG signaling plays a role in the prevention of the mPTP opening by inhibition of phosphodiesterases and is implicated in the mechanism underlying the cardioprotective effects of ANP. Therefore, it is reasonable to hypothesize that ANP may induce the cardioprotective effects by modulating the mPTP opening at reperfusion.Activation of several cardioprotective signaling pathways leads to prevention of the mPTP opening through glycogen synthase kinase3[3(GSK-3β) inactivation. Although little is known how ANP regulates GSK-3β activity in the heart, it is likely that ANP may prevent the mPTP opening by inactivating GSK-3β via PKG since1-methyl-3-isobutylxanthine (IBMX), a non-selective phosphodiesterases inhibitor, can modulate the mPTP opening through inactivation of GSK-3β via the cGMP/PKG pathway. In addition, the PI3K/Akt pathway may also be involved in the regulatory action of ANP on GSK-3(3activity since the PI3K/Akt is one of the important upstream signals that negatively regulate GSK-3β activity and ANP has been proposed to inhibite apoptosis by activating Akt.In the present study, we tested if ANP could modulate oxidative stress-induced mPTP opening through inactivation of GSK-3β in cardiac H9c2cells. We then investigated the signaling mechanism by which ANP inactivates GSK-3β.Results of the present study were showed as follow:1. Isolated rat hearts were perfused on a Langendorff apparatus and were subjected to30min regional ischemia followed by2h of reperfusion. ANP was gived for5min before reperfusion. ANP significantly reduced myocardial infarct size (P<0.001vs control), suggesting that ANP may prevent the heart from reperfusion injury.2. ANP significantly prevented loss of△Ψm caused by H2O2in a dose-dependent manner. The results suggest that ANP may modulate the mPTP opening (P<0.05, P<0.01vs control respectively).3. Western blotting results showed that ANP (0.Q1,0.1,1.0,10.0,100.0nmol/L) significantly increased phosphorylation of GSK-3β Ser9in H9c2cells (P<0.05, P<0.01vs control). However, ANP was not able to preserve TMRE fluorescence in cells transfected with GSK-3(3-S9A, indicating that ANP prevents the mPTP bpening by inactivating GSK-3β.4. The effects of ANP on GSK-3(3phosphorylation and△Ψm were reversed by the selective PKG inhibitor KT5823, implying that PKG mediates the inhibitory effect of ANP on GSK-3β. In support, the cGMP analogue8-Br-cGMP alone did not but together with PKG markedly enhanced GSK-3β phosphorylation (P<0.05vs ANP alone).5. ANP-induced GSK-3P phosphorylation was also abolished by the PI3K inhibitor LY294002and ANP could not prevent H2O2-induced loss of△Ψm in the presence of LY294002(P<0.05vs ANP alone). These results indicate that:1. ANP may prevent reperfusion injury in isolated rat heart by modulating the mPPT opening.2. ANP modulates oxidative stress-induced mPTP opening by inactivating GSK-3β via the PKG and PI3K/Akt signaling pathways.