Stimulation of ANP secretion by 2-Cl-IB-MECA through A(3) receptor and CaMKII

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.     

Attenuation of negatively regulated ANP secretion by calcium in hypertrophied atria

Abnormal intracellular Ca(2+)-handling has been described in various heart diseases associated with cardiac hypertrophy. The crucial role of Ca(2+) in the excitation-secretion coupling in atrial cardiomyocytes is not well established. To investigate modulation of atrial natriuretic peptide (ANP) secretion regulated by Ca(2+) in hypertrophied atria, responsiveness of stretch-induced ANP to Ca(2+) was studied using isolated perfused quiescent hypertrophied rat atria. Male Sprague-Dawley rats were given a single subcutaneous injection of 50 mg/kg monocrotaline (MCT) and were sacrificed at 5-6 weeks. In isolated perfused hypertrophied right atria from MCT rats, changes in atrial volume induced by increased atrial pressure caused proportional increases in mechanically stimulated extracellular fluid (ECF) translocation and stretch-induced ANP secretion. Stretch-induced ANP secretion was markedly increased by the depletion of extracellular Ca(2+). However, an accentuation of stretch-induced ANP secretion by Ca(2+) depletion was markedly attenuated in hypertrophied right atria, as compared to control right atria. Therefore, stretch-induced ANP secretion in terms of ECF translocation by Ca(2+) depletion in hypertrophied atria was significantly lower than in control right atria. However, no significant differences were observed between nonhypertrophied and control left atria. Depletion of extracellular Ca(2+) caused a decrease in intracellular calcium in single beating atrial myocytes, which was significantly attenuated in hypertrophied atrial myocytes. The results suggest that attenuation of Ca(2+)-induced negative regulation of ANP secretion in hypertrophied atria may be due to the disturbance of intracellular Ca(2+) regulation.     

Augmentation of moxonidine-induced increase in ANP release by atrial hypertrophy

Imidazoline receptors are divided into I(1) and I(2) subtypes. I(1)-imidazoline receptors are distributed in the heart and are upregulated during hypertension or heart failure. The aim of this study was to define the possible role of I(1)-imidazoline receptors in the regulation of atrial natriuretic peptide (ANP) release in hypertrophied atria. Experiments were performed on isolated, perfused, hypertrophied atria from remnant-kidney hypertensive rats. The relatively selective I(1)-imidazoline receptor agonist moxonidine caused a decrease in pulse pressure. Moxonidine (3, 10, and 30 micromol/l) also caused dose-dependent increases in ANP secretion, but clonidine (an alpha(2)-adrenoceptor agonist) did not. Pretreatment with efaroxan (a selective I(1)-imidazoline receptor antagonist) or rauwolscine (a selective alpha(2)-adrenoceptor antagonist) inhibited the moxonidine-induced increases in ANP secretion and interstitial ANP concentration and decrease in pulse pressure. However, the antagonistic effect of efaroxan on moxonidine-induced ANP secretion was greater than that of rauwolscine. Neither efaroxan nor rauwolscine alone has any significant effects on ANP secretion and pulse pressure. In hypertrophied atria, the moxonidine-induced increase in ANP secretion and decrease in pulse pressure were markedly augmented compared with nonhypertrophied atria, and the relative change in ANP secretion by moxonidine was positively correlated to atrial hypertrophy. The accentuation by moxonidine of ANP secretion was attenuated by efaroxan but not by rauwolscine. These results show that moxonidine increases ANP release through (preferentially) the activation of atrial I(1)-imidazoline receptors and also via different mechanisms from clonidine, and this effect is augmented in hypertrophied atria. Therefore, we suggest that cardiac I(1)-imidazoline receptors play an important role in the regulation of blood pressure.     

Attenuation of hypoosmotic stress-induced ANP secretion via I(Cl,swell) in renal hypertensive rat atria

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.     

Diadenosine tetraphosphate stimulates atrial ANP release via A(1) receptor: involvement of K(ATP) channel and PKC

Diadenosine polyphosphates (APnAs) are endogenous compounds and exert diverse cardiovascular functions. However, the effects of APnAs on atrial ANP release and contractility have not been studied. In this study, the effects of diadenosine tetraphosphate (AP4A) on atrial ANP release and contractility, and their mechanisms were studied using isolated perfused rat atria. Treatment of atria with AP4A resulted in decreases in atrial contractility and extracellular fluid (ECF) translocation whereas ANP secretion and cAMP levels in perfusate were increased in a dose-dependent manner. These effects of AP4A were attenuated by A(1) receptor antagonist but not by A(2A) or A(3) receptor antagonist. Other purinoceptor antagonists also did not show any effects on AP4A-induced ANF release and contractility. The increment of ANP release and negative inotropy induced by AP4A was similar to those induced by AP3A, AP5A, and AP6A. Protein kinase A inhibitors accentuated AP4A-induced ANP secretion. In contrast, an inhibitor of phospholipase C, protein kinase C or sarcolemma K(ATP) channel completely blocked AP4A-induced ANP secretion. However, an inhibitor of adenylyl cyclase or mitochondria K(ATP) channel had no significant modification of AP4A effects. These results suggest that AP4A regulates atrial inotropy and ANP release mainly through A(1) receptor signaling involving phospholipase C-protein kinase C and sarcolemmal K(ATP) channel and that protein kinase A negatively modulates the effects of AP4A.     

Role of COX-1 and COX-2 on skin PGs biosynthesis by mechanical scratching in mice

We examined the involvement of cyclooxygenase (COX)-1 and COX-2 on mechanical scratching-induced prostaglandins (PGs) production in the skin of mice. The dorsal regions of mice were scratched using a stainless brush. COXs expressions in the skin were analyzed using real-time PCR and Western blotting. The effect of acetylsalicylic acid (ASA) on the ability of PGs production were determined based on skin PGs level induced by arachidonic acid (AA) application. Mechanical scratching increased PGD2, PGE2, PGI2 and PGF(2 alpha). COX-1 was constitutively expressed and COX-2 expression was enhanced by scratching. Intravenous administration of ASA inhibited PGs biosynthesis in the normal skin. PGs levels of the skin 6h after ASA administration (ASA 6 h) were almost equal to those of the skin 10 min after ASA administration (ASA 10 min). In the scratched skin, AA-induced PGE2 and PGI2 of ASA 6 h were significantly higher than those of ASA 10 min. The skin PGD2 and PGF(2 alpha) of ASA 10 min were almost same to those of ASA 6 h. In the normal skin of COX-1-deficient mice, skin PGD2 level was lower than that of wild-type mice, although PGE2, PGI2 and PGF(2 alpha) levels were almost equal to those of wild type. In the scratched skin of COX-1-deficient mice, PGD2, PGE2, PGI2 and PGF(2 alpha) levels were lower than those of wild-type mice. These results suggested that cutaneous PGD2 could be mainly produced by COX-1, and PGE2 and PGI2 could be produced by COX-1 and COX-2, respectively, in mice.     

Angiotensin-(1-5), an active mediator of renin-angiotensin system,stimulates ANP secretion via Mas receptor

Angiotensin-(1-5) [Ang-(1-5)], which is a metabolite of Angiotensin-(1-7) [Ang-(1-7)] catalyzed by angiotensin-converting enzyme (ACE), is a pentapeptide of the renin-angiotensin system (RAS). It has been reported that Ang-(1-7) and Ang-(1-9) stimulate the secretion of atrial natriuretic peptide (ANP) via Mas receptor (Mas R) and Ang II type 2 receptor (AT₂R), respectively. However, it still remains unknown whether Ang-(1-5) has a similar function to Ang-(1-7). We investigated the effect of Ang-(1-5) on ANP secretion and to define its signaling pathway using isolated perfused beating rat atria. Ang-(1-5) (0.3, 3, 10 μM) stimulated high pacing frequency-induced ANP secretion in a dose-dependent manner. Ang-(1-5)-induced ANP secretion (3 μM) was attenuated by the pretreatment with an antagonist of Mas R (A-779) but not by an antagonist of AT₁R (losartan) or AT₂R (PD123,319). An inhibitor for phosphatidylinositol 3-kinase (PI3K; wortmannin), protein kinase B (Akt; API-2), or nitric oxide synthase (NOS; L-NAME) also attenuated the augmentation of ANP secretion induced by Ang-(1-5). Ang-(1-5)-induced ANP secretion was markedly attenuated in isoproterenol-treated hypertrophied atria. The secretagogue effect of Ang-(1-5) on ANP secretion was similar to those induced by Ang-(1-9) and Ang-(1-7). These results suggest that Ang-(1-5) is an active mediator of renin-angiotensin system to stimulate ANP secretion via Mas R and PI3K-Akt-NOS pathway.     

Suppression of high pacing-induced ANP secretion by antioxidants in isolated rat atria

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. H₂O₂ 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.     

Regulation of stretch-activated ANP secretion by chloride channels

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.     

Suppression of high pacing-induced ANP secretion by antioxidants in isolated rat atria

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. H₂O₂ 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.     

Suppression of ANP secretion by somatostatin through somatostatin receptor type 2

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.     

Caveolae are essential for angiotensin II type 1 receptor-mediated ANP secretion

Caveolae may act as mechanosensors and function as binding sites for calcium ions. The intracaveolar localization of atrial natriuretic peptide (ANP) derived from the direct interaction of atrial granules with caveolae has been demonstrated. The aim of this study was to define the effect of caveolae on ANP secretion induced by stretch and angiotensin II. The isolated perfused beating atria from Sprague-Dawley rats were used. To disrupt caveolae, 10 mM methyl-β-cyclodextrin (MbCD) was applied for 1 h and the number of caveoli were markedly decreased. MbCD increased basal ANP secretion and atrial diastolic pressure. The molecular profile of ANP in perfusate from control atria showed mainly one major peak corresponded to synthetic ANP whereas that from MbCD-treated atria showed two major immunoreactive peaks corresponded to synthetic rat ANP and proANP. High atrial stretch induced by elevating the height of outflow catheter from 5 cm H₂O to 7.5 cm H₂O increased atrial contractility and ANP secretion. The response of ANP secretion to high stretch was attenuated in MbCD-pretreated atria. Pretreatment with MbCD abolished angiotensin II-induced suppression and losartan-induced stimulation of ANP secretion. However, the effect of angiotenisin (1-7) on ANP secretion was not altered by MbCD treatment. The expression of angiotensin II type 1 receptor protein was reduced by MbCD treatment. These data suggest that caveolae are essential for angiotensin II type 1 receptor-mediated ANP secretion and relate to the processing of proANP.     

Oxidative stress augments the secretion of atrial natriuretic peptide in isolated 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, 1 mM) or hydrogen peroxide (0.1, 1, 10, 30 mM) was perfused into atria paced at 1.2 Hz. 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; 1 mM) and cariporide (an inhibitor of the Na⁺/H⁺ exchanger; 1 μM) but negative inotropic effect was not changed. U120 (a MAPK^erk pathway inhibitor; 10 μM) 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 AT2 receptor agonist stimulates high stretch induced- ANP secretion via PI3K/NO/sGC/PKG/pathway

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 1 μM) 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 (0.1 μM) 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 10 min 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 10 min 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.     

Effects of selective and non-selective COX inhibitors on antigen-induced release of prostanoid mediators and bronchoconstriction in the isolated perfused and ventilated guinea pig lung

The contribution of cycloxygenase (COX)-1 and COX-2 in antigen-induced release of mediators and ensuing bronchoconstriction was investigated in the isolated perfused guinea pig lung (IPL). Antigen challenge with ovalbumin (OVA) of lungs from actively sensitised animals induced release of thromboxane (TX)A(2), prostaglandin (PG)D(2), PGF(2)(alpha), PGI(2) and PGE(2), measured in the lung effluent as immunoreactive TXB(2), PGD(2)-MOX, PGF(2)(alpha), 6-keto PGF(1)(alpha) and PGE(2), respectively. This release was abolished by the non-selective COX inhibitor flurbiprofen (10 microM). In contrast, neither the selective COX-1 inhibitor FR122047 nor the selective COX-2 inhibitor celecoxib (10 microM each) significantly inhibited the OVA-induced bronchoconstriction or release of COX products, except for PGD(2). Another non-selective COX inhibitor, diclofenac (10 microM) also significantly inhibited antigen-induced bronchoconstriction. The data suggest that both COX isoenzymes, COX-1 and COX-2 contribute to the immediate antigen-induced generation of prostanoids in IPL and that the COX-1 and COX-2 activities are not associated with different profiles of prostanoid end products.     

C-type natriuretic peptide inhibits ANP secretion and atrial dynamics in perfused atria: NPR-B-cGMP signaling

The purpose of the present experiments was to define the role of C-type natriuretic peptide (CNP) in the regulation of atrial secretion of atrial natriuretic peptide (ANP) and atrial stroke volume. Experiments were performed in perfused beating and nonbeating quiescent atria, single atrial myocytes, and atrial membranes. CNP suppressed in a dose-related fashion the increase in atrial stroke volume and ANP secretion induced by atrial pacing. CNP caused a right shift in the positive relationships between changes in the secretion of ANP and atrial stroke volume or translocation of the extracellular fluid (ECF), which indicates the suppression of atrial myocytic release of ANP into the paracellular space. The effects of CNP on the secretion and contraction were mimicked by 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP). CNP increased cGMP production in the perfused atria, and the effects of CNP on the secretion of ANP and atrial dynamics were accentuated by pretreatment with an inhibitor of cGMP phosphodiesterase, zaprinast. An inhibitor of the biological natriuretic peptide receptor (NPR), HS-142-1, attenuated the effects of CNP. The suppression of ANP secretion by CNP and 8-BrcGMP was abolished by a depletion of extracellular Ca(2+) in nonbeating atria. Natriuretic peptides increased cGMP production in atrial membranes with a rank order of potency of CNP > BNP > ANP, and the effect was inhibited by HS-142-1. CNP and 8-BrcGMP increased intracellular Ca(2+) concentration transients in single atrial myocytes, and mRNAs for CNP and NPR-B were expressed in the rabbit atrium. From these results we conclude that atrial ANP release and stroke volume are controlled by CNP via NPR-B-cGMP mediated signaling, which may in turn act via regulation of intracellular Ca(2+).     

Endogenous angiotensin II suppresses stretch-induced ANP secretion via AT1 receptor pathway

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 5 cmH₂O to 7.5 cmH₂O. 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.     

Expression of enzymes of cyclooxygenase pathway and secretion of prostaglandin E2 and F2alpha by porcine myometrium during luteolysis and early pregnancy

Past studies of uterine prostaglandin (PGs) and pig reproduction have focused on endometrial rather than myometrial PGs. This study documents the synthesis and secretion of myometrial prostaglandins (PGs) in pigs and the involvement of oxytocin (OT) in these processes. Cyclooxygenase-2 (COX-2) expression was similar in myometrial explants from cyclic and pregnant pigs (days 14-16) and OT (10(-7) M) in vitro significantly increased COX-2 protein regardless of reproductive state. Basal expression of prostaglandin E2 synthase (PGES) was higher during pregnancy than during luteolysis. Conversely, prostaglandin F synthase (PGFS) was highest during luteolysis and lower in myometrium from gravid animals. OT had no influence on the expression of PGES and PGFS. In another tissue culture experiment, myometrial slices produced more PGE2 than PGF2alpha regardless of reproductive state of the female. OT stimulated PGE2 production in myometrium harvested during luteolysis and increased PGF2alpha production in all tissues examined. Progesterone (P4; 10(-5) M) blocked stimulatory effect of OT on myometrial PG release. Myometrial OTr mRNA was higher (P=0.03) during luteolysis than during pregnancy. In conclusion: (1) oxytocin increases myometrial COX-2 expression, but does not influence the expression of terminal enzymes of PGs synthesis (PGES and PGFS); (2) porcine myometrium preferentially produces PGs during early pregnancy and secretes more PGE2 than PGF2alpha; (3) myometrial OT and OTr support secretion of PGs from myometrium during luteolysis.     

Angiotensin III stimulates high stretch-induced ANP secretion via angiotensin type 2 receptor

Angiotensin III (Ang III) is metabolized from Ang II by aminopeptidase (AP) A and in turn, Ang III is metabolized to Ang IV by APN. Ang III is known to have a similar effect to Ang II on aldosterone secretion, but the effect of Ang III on atrial natriuretic peptide (ANP) secretion from cardiac atria is not known. The aim of the present study is to define the effect of Ang III on ANP secretion and its receptor subtype using isolated perfused beating atria. The volume load was achieved by elevating the height of outflow catheter connected with isolated atria from 5 cmH₂O to 7.5 cmH₂O. Atrial stretch by volume load increased atrial contractility and ANP secretion. Ang III stimulated stretch-induced ANP secretion in a dose-dependent manner without change in atrial contractility. The stimulated effect of Ang III (1 μM) on stretch-induced ANP secretion was blocked by the pretreatment of Ang II type 2 (AT₂) receptor antagonist but not by AT₁ or Mas receptor antagonist. Pretreatment with inhibitor of phosphoinositide 3-kinase (PI3K), Akt, nitric oxide synthase, soluble guanylyl cyclase, or protein kinase G (PKG) attenuated Ang III-stimulated ANP secretion. When Ang III (40 nM) or Ang II (4 nM) was infused for 10 min into anesthetized rats, mean arterial pressure was increased about 10%. However, Ang III increased plasma ANP level by 35.81 ± 10.19% but Ang II decreased plasma ANP level by 30.41 ± 7.27%. Therefore, we suggest that Ang III, opposite to Ang II, stimulated stretch-induced ANP secretion through AT₂ receptor/PI3K/Akt/nitric oxide/PKG pathway.