Increased ANP release under hypoxia is an adaptive biological change of atrium during the pathogenesis of cardiopulmonary diseases and it is more relevant to understand what underlying molecular mechanism is involved to explore the effective therapeutic target for disease. The present study focused on PTKs and found that its antagonist, genistein, are the key regulators of acute hypoxia-induced ANP secretion in the isolated perfused beating rat atria, with a potential downstream signaling pathway of PTKs-PI3K/Akt-HIF-1α pathway.
PTKs are important enzymes that catalyze substrate protein phosphorylation, where they transmit extracellular signals to the intracellular space and activate various intracellular signaling pathways that regulate processes of cell growth, differentiation, metabolism, migration, and apoptosis . Also, studies suggested that they control the abnormal signaling transduction of cells under ischemia conditions [25–27]. As reported in previous studies, hypoxia and ischemia are a strong stimulus for ANP release from cells or tissue . Herein, we focus on the functional involvement of PTK by observing the role of its antagonist genistein in hypoxia-induced ANP process in rat isolated perfused beating atria. Our data suggested that genistein effectively suppressed hypoxia-induced ANP section without influence on atrial pressure suggesting the negative role of genistein in hypoxia-induced ANP secretion.
Hypoxia-response elements have been characterized from the promoter sequence of NPs genes, suggesting that NPs genes are under controlled by HIF. That has been demonstrated to be involved in regulating the expression of various hypoxia-dependent genes. It has been demonstrated that PTKs are involved in the regulation of HIF-1α protein expression under hypoxia, and modulate HIF-1α activity through the PI3K/Akt signaling pathway . Results of our study also show that the PTK inhibitor, genistein decreased phosphorylation level of Akt and reduced hypoxia-induced HIF-1α protein expression in perfused rat atria. These data suggested that genistein inhibited PTK downstream PI3K/Akt signals. This was confirmed by present data of Akt agonist combined with genistein co-perfusion experiment which showed that activated Akt alleviated the inhibitory role of genistein on ANP secretion, HIF-1α expression and p-AKT expression. We thus examined this pathway in hypoxia-induced ANP section.
As the tissues adapt to hypoxic conditions, HIF-1α plays a key role in the regulation of expression and transcription of multiple hypoxia-related genes including PDGF, IGF-1, EGF, VEGF, and ANP [31, 32]. In a previous study, we have demonstrated that acute hypoxia significantly increases atrial HIF-1α activity and regulates hypoxia-induced ANP secretion . The present study showed that genistein alone greatly inhibited hypoxia-increased p-Akt and HIF-1α expressions, and significantly attenuated hypoxia-promoted atrial ANP secretion. However, genistein combined with IGF-1, an agonist for PI3K/Akt, treatment, not only markedly attenuated or completely blocked hypoxia-induced ANP secretion but also blocked the increase of hypoxia-induced HIF-1α. To determine the key role of HIF-1α in the inhibitory role of genistein on the ANP section, we performed rat atrial co-perfusion experiments with genistein plus rotenone or CAY10585 (two inhibitors for HIF-1α). The data showed that rotenone or CAY10585 did not alter the biological effect of genistein on hypoxia-induced ANP secretion. These data suggested that HIF-1α is an effective responder for genistein in reduced ANP secretion.
In conclusion, acute hypoxia significantly promotes ANP secretion in beating rat atria. Inhibition of PTKs by genistein regulates hypoxia-induced ANP secretion through PI3K/Akt-HIF-1α signaling pathways. HIF-1α and related hypoxia-induced signaling components adapt cells to hypoxic conditions and thus, the marked increase of ANP secretion is a functional behavior of atrial tissue under hypoxic stress.