Effect And Mechanisms Of Ouabain On Atrial Dynamics And ANP Secretion In Isolted Beating Rabbit Atria

Effect and mechanisms of ouabain on atrial dynamics in isolted beating rabbit atriaHeart failure is the weakening of cardiac myocytes contractility, leading to cardiac pump dysfunction. Contractility decline mainly due to the balance of cardiac myocytes calcium homeostasis is destroyed, followed with the intracellular calcium reducing and then calcium transient has been weakening. Heart failure is a syndrome, which heart diseases late performance together, is an important cause of cardiac death. Recently, the dominant drugs for the treatment of heart failure are digitalis classes and phosphodiesterases inhibitors (milrinone and amrinone). Ouabain, as an ibhibitor of the Na+-K+/ATPase (sodium pump), is one of the substances of the digitalis classes. Endogenous ouabain (EO) is a newly discovered steroid hormone, which is also the Na+-K+/ATPase inhibitor. It has important pathological and physiological significance. It has been demonstrated that EO secretion was affected by several fators such as levies of the salt uptake, blood volume, renin-angeotensin-aldosterone system, nervous system, insulin and hypoxia. EO is involved in the regualtion of body fluid, electrolyte balance and regulation of blood pressure. While exogenous ouabain, extracted from the seed of Strophanthus gratus Franchet of Acynaceae plant, is a fast, short-acting and potent cardiac glycoside. Na+-K+/ATPase hydrolyzes ATP to maintain the transmembrane gradients of Na+and K+found in most mammalian cells and is inhibited specifically by cardiac glycosides such as ouabain. Digitalis receptor is the Na+-K+/ATPase of plasma membranes. Inhibition of Na+-K+/ATPase by cardiac glycosides including ouabain has been shown to cause an increase in the concentration of intracellular Na+and lead to an increase in the concentration of intracellular Ca2+by activation of Na+/Ca2+exchangers, and finaly to produce a positive inotropic effect in the heart. This is the commen mechanisms by which Na+-K+/ATPase inhibitor, including digitalis cause a positive inotropic effect in the heart. There was reported that ouabain induced increased in cardiac contractility by activation of L-type Ca2+channels. In addition, Signal transducing function of Na+-K+/ATPase is essential for ouabain’s effect on [Ca2+]; in rat cardiac myocytes and it was recognized that activation of the Ras/MAPK pathway is necessary for the elevation of intracellular Ca2+by ouabain. On the other hand, several studies have been shown that Five minutes after application, low concentrations of ouabain induced Ca2+oscillations and stimulated ET-1release from endothelial cells into the medium and causes an elevation of vascular tone. Xavier et al reported that ouabain induced up regulation of ET-1gene and endothelin type A (ETA) receptor mRNA expression by treated with ouabain (8μg/d). In addition, it was demostrated that ouabain increased plasma and the heart ET levels and up regulated ETA mRNA expression in the left ventricular myocytes. It was also reported that ouabain may induced the positive inotropic effect in the papillary by activation of the endothelin type B (ETB) receptor in rabbits.Therefore, the purpose of the present study is to invetigate effect and mechanisms of ouabain, Na+-K+/ATPase inhibitor, on the atrial dynamics. These provide a theoretical basis for the role of ouabain on cardiac function as well as clinical diagnosis and treatment of the correlated diseasesThe results of the present study showed that,1.Atrial stroke volume and pulse pressure began to increase in the perfusion of ouabain (3.0μmol/L). These two reached peak after dealing by perfusion of ouabain for10minutes and12minutes respectively (compared with the control cycle for all P<0.001). The stroke volume and pulse pressure fell a little during the recovery period after stopping the perfusion, then rose gradually and reached its peak and to maintain a high level (with the control loop, both P<0.01). These results suggest that ouabain can enhance mechanical activities of rabbit atrial.2. KB-R7943(3.0μmol/L), an inhibitor of Na+/Ca2+exchanger, blocked the effect of ouabain-increased atrial stroke volume and failed to modulation of ouabain-induced increase in atrial pulse pressure. Nifedipine (3.0μmol/L), a blocker of L-type Ca2+channel, augmented ouabain-increased atrial stroke volume and the pulse pressure. There is a caution that ouabain furtherly increase the concentration of intracellular Ca+if nifedipine exists.3. A non-selective protein kinases inhibitor staurosporine (3.0μmol/L) can not affected the effect of ouabain-induced increase in atrial dynamics and this indicated that there was no correlations beween ouabain-increased atrial dynamics and protein kinases.4. High doses of ouabain (3.0and6.0μmol/L) significantly increased the secretion of ET-1in rabbit atria with a dose-dependent manner. Exogenous ET-1caused an increase of the atrial stroke volume in a dose-dependent manner.5. An ETA receptor antagonist BQ123(0.3μmol/L) blocked the effect of atrial stroke volume with significantly attenuated the increase in atrial pulse pressure induced by ouabain. ETB receptor blocker BQ788(0.3μmol/L) both significantly attenuated the effect of ouabain increased atrial pulse pressure and stroke volume in the recovery stage. The result suggests that the effect of ouabain enhancing mechanical activities of atrial is related to ET-1which is generated by atria.6. An inhibitor of MAPK PD98059(30.0μmol/L), completely blocked ouabain-promoted the generation of ET-1as well as ouabain-increased atrial dynamics. We can judge from that Ouabain enhance mechanical activities of atrial through the ET-1which is generated by the MAPK signal transduction pathway.7. Indomethacin (30.0μmol/L), an inhibitor of cyclooxiginase, completely blocked the effect of obubain-enhanced mechanical activities of atria, which indicates prostaglandin was also involved in the mechanisms on ouabain-induced changes of the atrial dunamics.The results of the present study indicate that,1. Ouabain activates Na+-Ca2+exchanger through inhibiting Na+-K+/ATPase to enhance the mechanical activities of the rabbit atrium;2. ET-1and MAPK signaling pathway revealed important roles in ouabain-increased atrial dynamics in isolated perfused beating rabbit atria.. Effect and the mechanisms of ouabain on atrial natriuretic peptide secretion in the perfused beating rabbit atriaThe heart as an endocrine gland generates and secretes natriuretic peptides (NPs) and to regulates body fluid as well as blood pressure. Natriuretic peptides were stored in human and mammal as family of natriuretic peptides (NPs) hormon. There are four members in the NPs including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP) and dendroaspis natriuretic peptide (DNP). All of the NPs members have been involved in the balancing of water and salt, regulation of blood pressure, diuresis and natriuresis, anti-proproliferation, regulation of the immunologic function, participating lipolysis, antioxidant and affecting motility of sperm.ANP is the first member of the NPs that it was synthesized and stored in atrial myocytes. It is demonstrated that many factors have been involved in the regulation of atrial ANP secretion, such as stretch of the atrial wall, endothelin-1(ET-1) and hypoxia while the atrial stretch has been considered to be a critical factor in the regulation of atrial ANP secretion. Therefore, any factors of affecting atrial stretch may involve in the regulation of ANP secretion.Ouabain has been recognized as an inhibitor of Na+-K+/ATPase. Endogenous ouabain, is one of the steroid hormone, secreted from adrenal cortex as well as hypothalamus. It has been demonstrated that EO secretion was affected by several factors such as levles of the salt uptake, blood volume, renin-angeotensin-aldosterone system, nervous system, insulin and hypoxia. EO is involved in the regualtion of body fluid, electrolyte balance and regulation of blood pressure. Exogenous ouabain, a digitalis species, has been extracted from bitter strophanthin seed of apocynaceae plant. Ouabain also has the inhibitory function of Na+-K+/ATPase in several tissues. Several studies demonstrated that ouabain may causes a significantly increase in atrial ANP secretion. However, the mechanisms by which ouabain increased atrial ANP secretion remains to be defined.Therefore, the purpose of the present study is to investigate the effect and mechanisms of ouabain on atrial ANP secretion by radioimmunoassay, physiological and pharmacological techniques in isolated perfused beating rabbit atria.The results of the present study showed that,1. The control period (12min) was followed by an infusion of ouabain (12min) and continued two cycles of the recovery (24min, perfused normal buffer without contained of ouabain). Ouabain (1.0,3.0, and6.0μmol/L) significantly increased atrial ANP secretion in the recovery period (at3.0and6.0μmol/L of ouabain, P<0.01and.P<0.001vs control respectively) in a dose-dependent manner but not in ouabain perfusion period.2. L-type Ca2+channel blocker nifedipine (3.0μmol/L) completely abolished the effect of ouabain-induced ANP secretion in the recovery period (P>0.05vs nifedipine alone). KB-R7943(3.0μmol/L), an inhibitor of Na+-Ca2+exchanger, failed to modulates of the effect of ouabain-induced increase in atrial ANP secretion (P<0.001vs KB-R7943alone).3. Ouabain (3.0and6.0μmol/L) significantly increased atrial ET-1secretion (P<0.05P<0.01vs control) in a dose-dependent manner. Exogenous ET-1(3.0,30.0and300.0nmol/L) significantly increased ANP secretion and showed a dose-dependent manner (P<0.05, P<0.01and P<0.001vs control respectively).4. The ETBreceptor antagonist BQ788(0.3μmol/L) completely blocked the effect of ET-1increased ANP secretion, but the ETA receptor blocker BQ123(0.3μmol/L) attenuated the effect of ET-1increased ANP secretion only.5. BQ788(0.μmol/L) completely blocked the effect of ouabain-increased ANP secretion in the recovery period (P<0.001vs ouabain alone) but not by BQ123. These results suggest that ouabain increase the atrial ANP secretion mainly via activation of ETB recptor. 6. MAPK inhibotr PD98059(30.0μmol/L) completely blocked the effect of ouabain increased ET-1production as well as the atrial ANP secretion. These results indicate that MAPK signaling pathway is involved in the ouabain-induced ET-1production and the atrial ANP secretion.7. Cyclooxygenase inhibitor indomethacin (30.0μmol/L) also completely blocked the effect of ouabain-increased ANP secretion in the recovery period and, therefore, it is indicates that prostaglandin formation caused by ouabain is involved in the ouabain-increased atrial ANP secretion.The results of the present study indicate that,1. Ouabain significantly increased atrial ANP secretion via ET-1-ETB receptor mediated pathway.2. MAPK activation and the prostaglandin formation caused by ouabain may play important roles in the ouabain-increased atrial ET-1as well as the ANP secretion in beating rabbit atria.