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Reactive oxygen species (ROS) are formed as a natural by-product of the normal metabolism of oxygen and have important roles in cell signaling. The aim of this study was to investigate direct effects of ROS on atrial hemodynamics and ANP secretion in isolated perfused beating rat atria with antioxidants. When atria were paced at 1.2 Hz, N-acetyl cystein (antioxidant, NAC), α-lipoic acid (antioxidant), tempol (superoxide dismutase mimic), and apocynin (NADPH oxidase inhibitor; NOX inhibitor) did not affect ANP secretion and atrial contractility. When pacing frequency was increased from 1.2 Hz to 4 Hz, the ANP secretion increased and atrial contractility decreased. H(2)O(2) level was increased in perfusate obtained from atria stimulated by high pacing frequency. NAC, α-lipoic acid and tempol attenuated high pacing frequency-induced ANP secretion but apocynin did not. In contrast, pyrogallol (a superoxide generator) augmented high pacing frequency-induced ANP secretion. NOX-4 protein was increased by high pacing stimulation and in diabetic rat atria. In diabetic rat atria, high pacing frequency caused an increased ANP secretion and a decreased atrial contractility, that were markedly attenuated as compared to control rats. NAC and apocynin reduced high pacing frequency-induced ANP secretion in diabetic rat atria. These results suggest that intracellular ROS formation partly through an increasing NOX activity in response to high pacing frequency is associated with an increased ANP secretion in rat atria.

Reactive oxygen species (ROS) play a role in cardiovascular diseases such as hypertension and heart failure. The objective of the present study was to investigate the role of endogenous ROS in atrial hemodynamics and ANP secretion in isolated perfused beating rat atria. Pyrogallol (a generator of superoxide anion, 0.1, 1mM) or hydrogen peroxide (0.1, 1, 10, 30mM) was perfused into atria paced at 1.2Hz. Pyrogallol and hydrogen peroxide stimulated ANP secretion and concentration in a dose-dependent manner and dramatically decreased atrial contractility and translocation of extracellular fluid. The stimulatory effect of pyrogallol and hydrogen peroxide on ANP secretion was attenuated by the pretreatment with ascorbic acid (an antioxidant; 1mM) and cariporide (an inhibitor of the Na(+)/H(+) exchanger; 1μM) but negative inotropic effect was not changed. U120 (a MAPK(erk) pathway inhibitor; 10μM) attenuated the stimulatory effect of hydrogen peroxide on ANP secretion. However, U120 augmented negative inotropic effect and stimulatory effect of ANP concentration induced by pyrogallol. Antioxidant such as N-acetyl cystein, gallate, propyl gallate, or ellagic acid did not cause any significant changes in atrial parameters. These results suggest that intracellular - formed ROS stimulates ANP secretion partly through activation of MAPK(erk) pathway and Na(+)/H(+) exchanger.

Angiotensin II (Ang II) is released by stretch of cardiac myocytes and has paracrine and autocrine effects on cardiac myocytes and fibroblasts. However, the direct effect of Ang II on the secretion of atrial natriuretic peptide (ANP) is unclear. The aim of the present study is to test whether Ang II affects stretch-induced ANP secretion. The isolated perfused beating atria were used from control and two-kidney one-clip hypertensive (2K1C) rats. The volume load was achieved by elevating the height of outflow catheter connected with isolated atria from 5cmH(2)O to 7.5cmH(2)O. Atrial stretch by volume load caused increases in atrial contractility by 60% and in ANP secretion by 100%. Ang II suppressed stretch-induced ANP secretion and tended to increase atrial contractility whereas losartan stimulated stretch-induced ANP secretion. Neither PD123319 nor A779 had direct effect on stretch-induced ANP secretion. The suppressive effect of Ang II on stretch-induced ANP secretion was blocked by the pretreatment of losartan but not by the pretreatment of PD123319 or A779. In hypertrophied atria from 2K1C rats, stretch-induced ANP concentration attenuated and atrial contractility augmented. The response of stretch-induced ANP secretion to Ang II and losartan augmented. The expression of AT1 receptor protein and mRNA increased but AT2 and Mas receptor mRNA did not change in 2K1C rat atria. Therefore, we suggest that Ang II generated endogenously by atrial stretch suppresses stretch-induced ANP secretion through the AT1 receptor and alteration of Ang II effect in 2K1C rat may be due to upregulation of AT1 receptor.

Adenosine is a potent mediator of myocardial protection against hypertrophy via A(1) or A(3) receptors that may be partly related to atrial natriuretic peptide (ANP) release. However, little is known about the possible involvement of the A(3) receptor on ANP release. We studied the effects of the A(3) receptor on atrial functions and its modification in hypertrophied atria. A selective A(3) receptor agonist, 2-chloro-N(6)-(3-iodobenzyl) adenosine-5'-N-methyluronamide (2-CI-IB-MECA), was perfused into isolated, beating rat atria with and without receptor modifiers. 2-CI-IB-MECA dose-dependently increased the ANP secretion, which was blocked by the A(3) receptor antagonist, but the increased atrial contractility and decreased cAMP levels induced by 30muM 2-CI-IB-MECA were not affected. The 100muM 2-(1-hexylnyl)-N-methyladenosine (HEMADO) and N(6)-(3-iodobenzyl) adenosine-5'-N-methyluronamide (IB-MECA), A(3) receptor agonist, also stimulated the ANP secretion without positive inotropy. The potency for the stimulation of ANP secretion was 2-CI-IB-MECA>>IB-MECA=HEMADO. The inhibition of the ryanodine receptor or calcium/calmodulin-dependent kinase II (CaMKII) attenuated 2-CI-IB-MECA-induced ANP release, positive inotropy, and translocation of extracellular fluid. However, the inhibition of L-type Ca(2+) channels, sarcoplasmic reticulum Ca(2+)-reuptake, phospholipase C or inositol 1,4,5-triphosphate receptors did not affect these parameters. 2-CI-IB-MECA decreased cAMP level, which was blocked only with an inhibitor of CaMKII or adenylyl cyclase. These results suggest that 2-CI-IB-MECA increases the ANP secretion mainly via A(3) receptor activation and positive inotropy by intracellular Ca(2+) regulation via the ryanodine receptor and CaMKII.

Cardiac hypertrophy, an adaptive process to an increased hemodynamic overload, includes not only an increase in cell size but also qualitative changes in constituent proteins. Although swelling-activated chloride channels (I(Cl,swell)) chronically activate in hypertrophied atrial myocytes, the role of I(Cl,swell) in regulation of atrial natriuretic peptide (ANP) release is poorly understood. We investigated the effects of I(Cl,swell) on ANP release and contractility and its modification in hypertrophied rat atria. To stimulate I(Cl,swell), hypoosmotic HEPES buffered solution (0.8T, 0.7T and 0.6T) was perfused into isolated perfused beating atria. The hypoosmotic HEPES buffered solution increased ANP release as compared to isoosmotic buffered solution (1T) in an osmolarity-reduction dependent manner. Atrial contractility and extracellular fluid translocation did not change. Exposure to hypoosmotic buffer (0.8T) containing low chloride (8mM), tamoxifen or diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) significantly attenuated hypoosmolarity-induced ANP release. The pretreatment with genistein, okdaic acid, U73122, GF109203x, and staurosporine attenuated hypoosmolarity-induced ANP release whereas orthovanadate augmented it significantly. In hypertrophied atria from renal hypertensive rats, hypoosmolarity-induced ANP release was markedly attenuated and DIDS-induced decrease in ANP release and negative inotropy were augmented as compared to sham-operated rat atria. Therefore, we suggest that I(Cl,swell) may partly participate hypoosmolarity-induced ANP release through protein tyrosine kinase and phospholipase C-protein kinase C pathway. The modification of responses of ANP release to hypoosmolarity and DIDS in hypertrophied atria may relate to changes in I(Cl,swell) activity by persistent high blood pressure.

This study was aimed to define roles of stretch-activated ion channels (SACs), especially Cl(-) channels, in regulation of atrial natriuretic peptide (ANP) secretion using isolated perfused beating atria. The volume load was achieved by elevating height of outflow catheter connected to isolated rat atria and the pressure load was achieved by decreasing diameter of outflow catheter. Both methods increased atrial contractility similarly although volume load was different (736microl for volume load vs. 129microl for pressure load). Atrial stretch by volume load markedly increased ECF translocation and ANP secretion but the pressure load slightly increased. The ANP secretion was positively correlated to workload generated by volume or pressure load. Treatment of atria with gadolinium, a blocker for SACs, attenuated the ECF translocation and the ANP secretion induced by volume load. A blocker for Ca2+-activated Cl(-) channel, niflumic acid (NFA), accentuated the ANP secretion induced by volume load whereas a blocker for swelling-activated Cl(-) channel, diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), attenuated the ANP secretion. The ANP secretion of hypertrophied atria by volume load was markedly reduced and the augmented effect of NFA on volume load-induced ANP secretion was not observed. These results indicate that Cl(-) channels may differently regulate stretch-activated ANP secretion.

Angiotensin II (Ang II) type 1 receptor (AT1R) mediates the major cardiovascular effects of Ang II. However, the effects mediated via AT2R are still controversial. The aim of the present study is to define the effect of AT2R agonist CGP42112A (CGP) on high stretch-induced ANP secretion and its mechanism using in vitro and in vivo experiments. CGP (0.01, 0.1 and 1μM) stimulated high stretch-induced ANP secretion and concentration from isolated perfused rat atria. However, atrial contractility and the translocation of extracellular fluid did not change. The augmented effect of CGP (0.1μM) on high stretch-induced ANP secretion was attenuated by the pretreatment with AT2R antagonist or inhibitor for phosphoinositol 3-kinase (PI3K), nitric oxide (NO), soluble guanylyl cyclase (sGC), or protein kinase G (PKG). However, antagonist for AT1R or Mas receptor did not influence CGP-induced ANP secretion. In vivo study, acute infusion of CGP for 10min increased plasma ANP level without blood pressure change. In renal hypertensive rat atria, AT2R mRNA and protein levels were up-regulated and the response of plasma ANP level to CGP infusion in renal hypertensive rats augmented. The pretreatment with AT2R antagonist for 10min followed by CGP infusion attenuated an increased plasma ANP level induced by CGP. However, pretreatment with AT1R or Mas receptor antagonist unaffected CGP-induced increase in plasma ANP level. Therefore, we suggest that AT2R agonist CGP stimulates high stretch-induced ANP secretion through PI3K/NO/sGC/PKG pathway and these effects are augmented in renal hypertensive rats.

Urotensin II (UII) is a vasoactive peptide which is bound to a G protein-coupled receptor. UII and its receptor are upregulated in ischemic and chronic hypoxic myocardium, but the effect of UII on ischemic reperfusion (I/R) injury is still controversial. The aim of the present study was to investigate whether UII protects heart function against I/R injury. Global ischemia was performed using isolated perfused Langendorff hearts of Sprague-Dawley rats. Hearts were perfused with Krebs-Henseleit buffer for 20min pre-ischemic period followed by a 20min global ischemia and 50min reperfusion. Pretreatment with UII (10nM) for 10min increased recovery percentage of the post-ischemic left ventricular developed pressure and ±dp/dt, and decreased post-ischemic left ventricular end-diastolic pressure as compared with I/R group. UII decreased infarct size and an increased lactate dehydrogenase level during reperfusion. Cardioprotective effects of UII were attenuated by pretreatment with UII receptor antagonist. The hydrogen peroxide activity was increased in UII-treated heart before ischemia. The Mn-SOD, catalase, heme oxygenase-1 and Bcl-2 levels were increased, and the Bax and caspase-9 levels were decreased in UII-treated hearts. These results suggest that UII has cardioprotective effects against I/R injury partly through activating antioxidant enzymes and reactive oxygen species.

Somatostatin is a cyclic-14 amino acid peptide which mainly distributed in digestive system and brain. Somatostatin receptor (SSTR) is a G-protein coupled receptor and all five SSTR subtypes are expressed in cardiomyocytes. The aim of this study was to investigate the effect of somatostatin on atrial natriuretic peptide (ANP) secretion and its signaling pathway. Somatostatin (0.01 and 0.1 nM) decreased ANP secretion in isolated beating rat atrium in a dose-dependent manner. But atrial contractility and translocation of extracellular fluid were not changed. Somatostatin-induced decrease in ANP secretion was significantly attenuated by the pretreatment with CYN 154806 (SSTR type 2 antagonist; 0.1 μM), but not by BIM 23056 (SSTR type 5 antagonist; 0.1 μM) and urantide (urotensin II receptor antagonist; 0.1 μM). When pretreated with an agonist for SSTR type 2 (Seglitide, 0.1 nM) and SSTR type 5 (L 817818, 0.1 nM), only Seglitide reduced ANP secretion similar to that of somatostatin. The suppressive effect of somatostatin on ANP secretion was attenuated by the pretreatment with an inhibitor for adenylyl cyclase (MDL-12330A, 5 μM) or protein kinase A (KT 5720, 0.1 μM). In diabetic rat atria, the suppressive effect of somatostatin on ANP secretion and concentration was attenuated. Real time-PCR and western blot shows the decreased level of SSTR type 2 mRNA and protein in diabetic rat atria. These data suggest that somatostatin decreased ANP secretion through SSTR type 2 and an attenuation of suppressive effect of somatostatin on ANP secretion in diabetic rat atria is due to a down-regulation of SSTR type 2.

Gap junction channels facilitate chemical and electrical communication between adjacent cells. Gap junction protein, connexin (Cx), is expressed in the endothelial cells of vessels, glomerulus, and renin-secreting cells of the kidney. The purpose of this study was to investigate the role of Cx in renin release using Cx-overexpressing As 4.1 cells. The adenovirus-induced Cx overexpression was conducted by using recombinant adenovirus containing the cDNA encoding Cx37, Cx40, Cx43 (Ad-Cx), and beta-galactosidase (Ad-beta-gal). In 40-overexpressing cells, basal renin release increased in a time-dependent manner but it was significantly lower than that in Ad-beta-gal-treated cells. In Cx37- and Cx43-overexpressing cells, basal renin release was increased in a time-dependent manner, which was not different from control cells. 18-beta glycyrrhetinic acid (GA), a gap junction blocker, stimulated renin release dose-dependently and increased intracellular Ca(2+) in both Cx43-overexpressing cells and control cells. However, no significant differences were observed. An increase in renin release by 3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester, a putative antagonist of Ca(2+) release from intracellular sequestration sites, was also similar between two groups. These results suggest that Cx43 may unlikely alter the regulation of renin release and intracellular Ca(2+) by gap junction blocker in As 4.1 cells.

Urotensin II (U-II) and its receptor are coexpressed in the heart and show various cardiovascular functions. However, the relationship between U-II and cardiac hormone atrial natriuretic peptide (ANP) is still unknown. The aim of the present study is to test whether U-II affects ANP secretion using in vitro perfusion experiments and in vivo studies. Human U-II (hU-II) (10 −11 , 5 × 10 −11 , 10 −10 , 5 × 10 −10 M) stimulated ANP secretion from isolated perfused rat atria paced with high frequency (6.0 Hz). However, atrial contractility and translocation of extracellular fluid (ECF) did not change. An increase in ANP secretion by rat U-II was similar to that by hU-II; however, urotensin-related peptide showed no significant effect on ANP secretion. Pretreatment with urotensin receptor antagonist and inhibitor for phospholipase C (PLC), phosphoinositide 3-kinase (PI3K), or protein kinase C (PKC) attenuated hU-II-induced ANP secretion from atria paced with high frequency, but an inhibitor for inositol triphosphate did not. Intravenous infusion of hU-II at a dose of 2.5 μM for 20 min increased plasma ANP level, along with increased heart rate and pulse pressure in anesthetized rats. Therefore, we suggest that U-II stimulates high stimulation frequency-induced ANP secretion partly through the urotensin receptor and the PLC/PI3K/PKC pathway.

Cyclooxygenase (COX) is a key enzyme regulating the production of various prostaglandins (PGs) from arachidonic acid. Angiotensin II has been reported to be an important inflammatory mediator, which increases COX-2. The aim of this study was to determine the role of various PGs and COX-2 in the regulation of atrial natriuretic peptide (ANP) secretion. PGF2alpha and PGD2 caused dose-dependent increases in ANP release and intra-atrial pressure. The potency for the stimulation of ANP secretion by PGF2alpha was higher than that by PGD2. In contrast, PGE2, PGI2, PGJ2, and thromboxane A2 did not show any significant effects. The increases in intra-atrial pressure and ANP secretion induced by PGF2alpha and PGD2 were significantly attenuated by the pretreatment with an inhibitor of PGF2alpha receptor. By the pretreatment with an inhibitor for phospholipase C (PLC), inositol 3-phosphate (IP3) receptor, protein kinase C (PKC), or myosin light chain kinase (MLCK), PGF2alpha-mediated increase in ANP secretion and positive inotropy were attenuated. Inhibitor for COX-1 or COX-2 did not cause any significant effects on atrial parameters. In hypertrophied rat atria, PGF2alpha-induced positive inotropy and ANP secretion were markedly attenuated whereas COX-2 inhibitor stimulated ANP secretion. The expression of COX-2 increased and the expression of PGF2alpha receptor mRNA decreased in hypertrophied rat atria. These results suggest that PGF2alpha increased the ANP secretion and positive inotropy through PLC-IP3-PKC-MLCK pathway, and the modulation of ANP secretion by COX-2 inhibitor and PGF2alpha may partly relate to the development of renal hypertension.

Sympathetic nervous system and atrial natriuretic peptide (ANP) system play fundamental roles in the regulation of cardiovascular functions. Overactivity of sympathetic nervous system can lead into cardiovascular diseases such as heart failure and hypertension. The present study aimed to define which adrenergic receptors (ARs) affect atrial contractility and ANP release and to determine their modification in renal hypertensive rat atria. An alpha(1)-AR agonist, cirazoline increased ANP release with positive inotropism. These alpha(1)-AR agonist-mediated responses were attenuated by the alpha(1A)-AR antagonist, but not by the alpha(1B)- or alpha(1D)-AR antagonist. An alpha(2)-AR agonist, guanabenz and clonidine increased ANP release with negative inotropism and decreased cAMP level. The order of potency for the increased ANP release was cirazolinephenylephrine=guanabenzclonidine. In contrast, a beta-AR agonist, isoproterenol decreased ANP release with positive inotropism and these responses were blocked by the beta(1)-AR antagonist but not by the beta(2)-AR antagonist. The increased cAMP level by isoproterenol was suppressed by pretreatment with both beta(1)- and beta(2)-AR antagonists. In renal hypertensive rat atria, the effects of isoproterenol on atrial contractility, ANP release, and cAMP level were attenuated whereas the effect of cirazoline on ANP release was unaltered. Atrial beta(1)-AR mRNA level but not alpha(1A)-AR mRNA level was decreased in renal hypertensive rats. These findings suggest that alpha(1A)- and beta(1)-AR oppositely regulate atrial ANP release and that atrial beta(1)-AR expression/function is impaired in renal hypertensive rats.