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Abstract: The alteration in osmolarity challenges cell volume regulation, a vital element for cell survival. Hyposmolarity causes an increase in cell volume. Recently, it has been reported that the renin–angiotensin system (RAS) plays a role in cell volume regulation. We investigated the effect of angiotensin-(1–7) [Ang-(1–7)] on hyposmolarity-induced atrial natriuretic peptide (ANP) secretion in normal and diabetic (DM) rat atria and modulation of the effect of Ang-(1–7) by the Na+–K+ pump. Using isolated control rat atria, we observed that perfusion of hyposmotic solution into the atria increased ANP secretion. When Ang-(1–7) [0.1μM or 1μM] was perfused in a hyposmolar solution, it decreased the hyposmolarity-induced ANP secretion in a dose-dependent manner. This effect of Ang-(1–7) could be mediated by the Na+–K+ pump, since ouabain, an Na+–K+ pump inhibitor, significantly decreased the effect of Ang-(1–7) on hyposmolarity-induced ANP secretion. In contrast, Nω Nitro-l-arginine methyl ester hydrochloride (l-NAME) did not modify the effect of Ang-(1–7) on the hyposmolarity-induced ANP secretion. Interestingly, the ANP secretion was increased robustly by the perfusion of the hyposmolar solution in the DM atria, as compared to the control atria. However, the inhibitory effect of Ang-(1–7) on the hyposmolarity-induced ANP secretion was not observed in the DM atria. In the DM atria, atrial contractility was significantly increased. Taken together, we concluded that Ang-(1–7) attenuated hyposmolarity-induced ANP secretion via the Na+–K+ pump and a lack of Ang-(1–7) response in DM atria may partly relate to change in Na+–K+ pump activity. [ABSTRACT FROM AUTHOR]

We have previously demonstrated that cyclic ADP-ribose (cADPR) is a calcium signaling messenger in interleukin 8 (IL- 8)-induced lymphokine-activated killer (LAK) cells. In this study we examined the possibility that 1L-8 activates CD38 to produce another messenger, nicotinic acid adenine dinucleotide phosphate (NAADP), in LAK cells, and we showed that IL-8 induced NAADP formation after cADPR production. These calcium signaling messengers were not produced when LAK cells prepared from CD38 knock-out mice were treated with IL-8, indicating that the synthesis of both NAADP and cADPR is catalyzed by CD38 in LAK cells. Application of cADPR to LAK cells induced NAADP production, whereas NAADP failed to increase intracellular cADPR levels, confirming that the production of cADPR precedes that of NAADP in IL-8-treated LAK cells. Moreover, NAADP increased intracellular Ca2+ signaling as well as cell migration, which was completely blocked by bafilomycin A 1, suggesting that NAADP is generated in lysosome-related organelles after cADPR production. IL-8 or exogenous cADPR, but not NAADP, increased intracellular cAMP levels, cGMP analog, 8-(4-chlorophenylthio)-guanosine 3′,5′-cyclic monophosphate, increased both cADPR and NAADP production, whereas the cAMP analog, 8-(4-chlorophenylthio)-cAMP, increased only NAADP production, suggesting that cAMP is essential for IL-8-induced NAADP formation. Furthermore, activation of Rap 1, a downstream molecule of Epac, was required for IL-8-induced NAADP formation in LAK cells. Taken together, our data suggest that IL-8-induced NAADP production is mediated by CD38 activation through the actions ofcAMP/Epac/protein kinase A/Rap1 in LAK cells and that NAADP plays a key role in Ca2+ signaling of IL-8-induced LAK cell migration. [ABSTRACT FROM AUTHOR]

Abstract: Objectives: The aim of the present studywas to examine the question of whether the atrial natriuretic peptide (ANP) system is altered by endothelial nitric-oxide synthase (eNOS). Methods: Male eNOS-deficient mice (eNOS−/−) and wild type control mice (eNOS+/+, C57B1/6J) were used. Blood pressure was measured in anesthetized mice by tail cuff plethysmography and renal function was measured. Expression of ANP, natriuretic peptide receptor (NPR)-A, NPR-C, and tonicity-responsive enhancer binding protein (TonEBP) mRNA was determined by real-time PCR. Localization of 125I-ANP binding sites was measured using in vitro autoradiography. Results: In eNOS−/− mice, systolic blood pressure increased and left ventricular hypertrophy was observed. Urine volume and osmolarity did not change. Expression of ANP markedly increased in the heart and kidney of eNOS−/− mice. Expression of NPR-A and NPR-C increased in the heart and tended to increase in the kidney of eNOS−/− mice. In the renal medulla in particular, increased expression of NPR-C was more prominent. Expression of TonEBP mRNA was markedly decreased in the renal medulla, but not in the renal cortex. Maximum binding capacity (Bmax) of ANP and C-ANP increased in the renal medulla in eNOS−/− mice. Conclusion: These results suggest that the eNOS-NO system may be partly involved in regulation of ANP, NPR-A, -C, and TonEBP mRNA expression in the kidney. [Copyright &y& Elsevier]

Abstract: 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 β-galactosidase (Ad-β-gal). In 40-overexpressing cells, basal renin release increased in a time-dependent manner but it was significantly lower than that in Ad-β-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-β glycyrrhetinic acid (GA), a gap junction blocker, stimulated renin release dose-dependently and increased intracellular Ca2+ 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 Ca2+ 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 Ca2+ by gap junction blocker in As 4.1 cells. [Copyright &y& Elsevier]

Abstract: Reactive oxygen species are important mediators that exert a toxic effect during ischemia–reperfusion (I/R) injury of various organs. Sulforaphane is known to be an indirect antioxidant that acts by inducing Nrf2-dependent phase 2 enzymes. In this study, we investigated whether sulforaphane protects heart against I/R injury. Sprague–Dawley rats received sulforaphane (500μg/kg/day) or vehicle intraperitoneally for 3 days and global ischemia was performed using isolated perfused Langendorff hearts. Hearts were perfused with Krebs-bicarbonate buffer for 20min pre-ischemic period followed by a 20min global ischemia and 50min reperfusion. Treatment with sulforaphane inhibited an increase in the post-ischemic left ventricular end-diastolic pressure (LVEDP) and improved the post-ischemic left ventricular developed pressure (LVDP), ±dP/dt, and coronary flow as compared with the untreated control hearts. Pretreatment with 5-hydroxydecanoic acid (5-HD), a mitochondrial KATP channel blocker, for 10min before ischemia attenuated the improvement of LVEDP, LVDP, ±dP/dt, and coronary flow induced by sulforaphane. Sulforaphane markedly decreased the infarcted size and attenuated the increased lactate dehydrogenase level in effluent during reperfusion. Pretreatment with 5-HD also blocked these protective effects of sulforaphane. Post-ischemia increased the concentration of atrial natriuretic peptide in coronary effluent, which attenuated by sulforaphane treatment. Decreases on Mn-superoxide dismutase (SOD), catalase, and heme oxygenase-1 levels by I/R were increased by sulforaphane treatment and pretreatment of 5-HD blocked the sulforaphane effects. Increases in Bax and caspase-3 levels, and decrease in Bcl-2 level by I/R were attenuated by sulforaphane treatment. These results suggest that the protective effects of sulforaphane against I/R injury may be partly mediated through mitochondrial KATP channels and antioxidant pathway. [Copyright &y& Elsevier]

Abstract: 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 (6.0Hz). 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 2.5μM for 20min 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. [Copyright &y& Elsevier]

Abstract: Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) is an uncoupler of mitochondrial oxidative phosphorylation in eukaryotic cells. Here, we evaluated the in vitro effects of FCCP on the growth of Calu-6 lung cancer cells. FCCP inhibited the growth of Calu-6 cells with an IC50 of approximately 6.64±1.84μM at 72h, as shown by MTT. DNA flow cytometric analysis indicated that FCCP induced G1 phase arrest below 20μM of FCCP. Treatment with FCCP decreased the level of CDKs and cyclines in relation to G1 phase. In addition, FCCP not only increased the p27 level but also enhanced its binding with CDK4, which was associated with hypophosphorylation of Rb protein. While transfection of p27 siRNA inhibited G1 phase arrest in FCCP-treated cells, it did not enhance Rb phosphorylation. FCCP also efficiently induced apoptosis. The apoptotic process was accompanied with an increase in sub-G1 cells, annexin V staining cells, mitochondria membrane potential (MMP) loss and cleavage of PARP protein. All of the caspase inhibitors (caspase-3, -8, -9 and pan-caspase inhibitor) markedly rescued the Calu-6 cells from FCCP-induced cell death. However, knock down of p27 protein intensified FCCP-induced cell death. Moreover, FCCP induced the depletion of GSH content in Calu-6 cells, which was prevented by all of the caspase inhibitors. In summary, our results demonstrated that FCCP inhibits the growth of Calu-6 cells in vitro. The growth inhibitory effect of FCCP might be mediated by cell cycle arrest and apoptosis via decrease of CDKs and caspase activation, respectively. These findings now provide a better elucidation of the mechanisms involved in FCCP-induced growth inhibition in lung cancer. [Copyright &y& Elsevier]

Abstract: 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. PGF2α and PGD2 caused dose-dependent increases in ANP release and intra-atrial pressure. The potency for the stimulation of ANP secretion by PGF2α 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 PGF2α and PGD2 were significantly attenuated by the pretreatment with an inhibitor of PGF2α 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), PGF2α-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, PGF2α-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 PGF2α receptor mRNA decreased in hypertrophied rat atria. These results suggest that PGF2α increased the ANP secretion and positive inotropy through PLC–IP3–PKC–MLCK pathway, and the modulation of ANP secretion by COX-2 inhibitor and PGF2α may partly relate to the development of renal hypertension. [Copyright &y& Elsevier]

Abstract: Extracellular osmolarity is known as an important factor for the regulation of natriuretic peptide receptors (NPRs). We investigated the intra-renal osmoregulation of NPRs using renal medullectomized rats with bromoethylamine hydrobromide (BEA, 200mg/kg). The administration of BEA caused the decreased food intake and body weight. Water intake was decreased on the first day and then increased from the second day. Urine volume was persistently increased from the first day and free water clearance was also increased from the second day. Urinary excretions of sodium and potassium were decreased on the second day and then recovered to control level. Plasma levels of atrial natriuretic peptide (ANP) and Dendroaspis natriuretic peptide (DNP) in BEA-treated rats were not different from control rats. The inactive renin was increased. The maximum binding capacities of 125I-ANP as well as 125I-DNP decreased in glomeruli and medulla of BEA-treated rat kidneys but the binding affinity was not changed. In renal cortex, the gene expressions of ANP, NPR-A, and NPR-B were not changed but that of NPR-C decreased. In renal medulla, the gene expressions of NPR-A, -B, and -C decreased without change in ANP mRNA. Both renal medullary osmolarity and sodium concentration by BEA treatment were lower than those in control kidney. The cGMP concentrations in renal medulla and urine in BEA-treated rats were higher than those in control rats. These results suggest that the increased cGMP production may be partly involved in the decrease in NPRs mRNA expression and their binding capacities by BEA-induced medullectomy. [Copyright &y& Elsevier]

Abstract: Cold exposure induces hypertension and cardiac hypertrophy via sympathetic activation. The sympathetic nervous system is fundamentally important for the regulation of cardiac atrial natriuretic peptide (ANP) secretion. The present study aimed to define changes in ANP level with renal functions during cold exposure of rats. We also measured the direct effects of adrenergic stimulation on ANP secretion in cold-induced hypertensive rat atria. Sustained elevation of blood pressure and tachycardia were observed by 2-wk cold exposure. Cold exposure increased urine volume, UNaV, UKV and positive water balance. Atrial ANP content, its mRNA level, and plasma ANP concentration increased. Plasma norepinephrine level was increased but both α1A- and β1-adrenoceptor (AR) mRNA levels in atrium were decreased. In isolated perfused atria from cold-exposed rats, basal ANP secretion increased and pulse pressure decreased. Phenylephrine (α1-AR agonist)-induced stimulation of ANP secretion, and isoproterenol (β-AR agonist)-induced suppression of ANP secretion were significantly attenuated. These results suggest that an increased plasma and atrial ANP level by cold exposure may be a compensatory response to changes in hemodynamics and body fluid balance. The phenylephrine- and isoproterenol-induced attenuation of ANP secretion in cold-exposed rat atria may be due to the downregulation of α1A- and β1-adrenoceptors mRNA levels. [Copyright &y& Elsevier]

Abstract: Adenosine is a potent mediator of myocardial protection against hypertrophy via A1 or A3 receptors that may be partly related to atrial natriuretic peptide (ANP) release. However, little is known about the possible involvement of the A3 receptor on ANP release. We studied the effects of the A3 receptor on atrial functions and its modification in hypertrophied atria. A selective A3 receptor agonist, 2-chloro-N6-(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 A3 receptor antagonist, but the increased atrial contractility and decreased cAMP levels induced by 30μM 2-CI-IB-MECA were not affected. The 100μM 2-(1-hexylnyl)-N-methyladenosine (HEMADO) and N6-(3-iodobenzyl) adenosine-5′-N-methyluronamide (IB-MECA), A3 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 Ca2+ channels, sarcoplasmic reticulum Ca2+-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 A3 receptor activation and positive inotropy by intracellular Ca2+ regulation via the ryanodine receptor and CaMKII. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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 α1-AR agonist, cirazoline increased ANP release with positive inotropism. These α1-AR agonist-mediated responses were attenuated by the α1A-AR antagonist, but not by the α1B- or α1D-AR antagonist. An α2-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 cirazoline≫phenylephrine=guanabenz≫clonidine. In contrast, a β-AR agonist, isoproterenol decreased ANP release with positive inotropism and these responses were blocked by the β1-AR antagonist but not by the β2-AR antagonist. The increased cAMP level by isoproterenol was suppressed by pretreatment with both β1- and β2-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 β1-AR mRNA level but not α1A-AR mRNA level was decreased in renal hypertensive rats. These findings suggest that α1A- and β1-AR oppositely regulate atrial ANP release and that atrial β1-AR expression/function is impaired in renal hypertensive rats. [Copyright &y& Elsevier]

Abstract: 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 (736μl for volume load vs. 129μl 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. [Copyright &y& Elsevier]

Abstract: 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 A1 receptor antagonist but not by A2A or A3 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 KATP channel completely blocked AP4A-induced ANP secretion. However, an inhibitor of adenylyl cyclase or mitochondria KATP channel had no significant modification of AP4A effects. These results suggest that AP4A regulates atrial inotropy and ANP release mainly through A1 receptor signaling involving phospholipase C-protein kinase C and sarcolemmal KATP channel and that protein kinase A negatively modulates the effects of AP4A. [Copyright &y& Elsevier]

Abstract: Antimycin A, an inhibitor of electron transport in mitochondria, has been used as reactive oxygen species (ROS) generator in the biological system. Here, we investigated the in vitro effect of antimycin A on apoptosis in As4.1 juxtaglomerular cells. Antimycin A efficiently induced apoptosis in As4.1 cells as evidenced by flow cytometric detection of sub-G1 DNA content, annexin V binding assay and DAPI staining. This apoptotic process was accompanied by loss of mitochondrial transmembrane potential (ΔΨ m), Bcl-2 decrease, caspase-3 activation and PARP cleavage. All of caspase inhibitors tested in this experiment failed to rescue As4.1 cells from antimycin A-induced cell death at the time of 48h in view of sub-G1 cells and annexin V positive staining cells. However, with regard to the mitochondrial membrane potential (ΔΨ m), pan caspase inhibitor (Z-VAD-FMK) and caspase-3 inhibitor (Z-DEVD-FMK) at the concentration of 25μM noticeably decreased the loss of mitochondrial membrane potential (ΔΨ m) in antimycin A-treated cells. Taken together, we have demonstrated that antimycin A as an inhibitor of electron transport in mitochondria potently induces apoptosis in As4.1 juxtaglomerular cells. [Copyright &y& Elsevier]

Abstract: The aim of present study was to define the effects of insulin on atrial dynamics and ANP release and its modification in diabetic rats. An isolated perfused beating atrial model was used from control and diabetic rats. Insulin was perfused with and without an inhibitor for tyrosine kinase or phosphatidylinositol 3-kinase (PI 3-kinase). Insulin increased the release of ANP and decreased atrial contractility in a dose-dependent manner. During the perfusion of 10−10 M insulin, the release of ANP abruptly increased within 8min by approximately 40% and then decreased with time despite of continuous perfusion. In terms of increasing the dose of insulin, the time to reach the peak effect became faster and the slope to decrease became slower. In contrast, atrial contractility was gradually decreased with time. These effects were independent upon extracellular glucose. Genistein (10−5 M) or lavendustin C (10−5 M), a tyrosine kinase inhibitor, attenuated the release of ANP stimulated by insulin (10−8 M). Wortmannin (10−7 M) or LY294002 (10−5 M), a PI 3-kinase inhibitor, also attenuated insulin-stimulated ANP release. However, both inhibitors for PI 3-kinase and tyrosine kinase did not cause any significant effects on negative inotropism by insulin. Insulin-stimulated ANP release was augmented in streptozotocin-treated rat atria. The density of insulin receptor markedly increased in diabetic hearts. These results suggest that insulin stimulates the release of ANP through PI 3-kinase and tyrosine kinase, and augmentation of insulin-stimulated ANP release in diabetic rat atria may be partly due to an upregulation of insulin receptor. [Copyright &y& Elsevier]

Dendroaspis natriuretic peptide (DNP), a 38-amino-acid peptide, was isolated from the venom of Green Mamba. It has structural and functional similarities to other members of the natriuretic peptide family. The purpose of this study was to determine whether DNP system is present in the rat colon and to define its biological functions. The serial dilution curve of extracts of colonic tissues was parallel to the standard curve of DNP and a major peak of molecular profile by HPLC was synthetic DNP. The concentration of DNP was 0.5±0.04 ng/g of colonic tissues. DNP as well as atrial natriuretic peptide and C-type natriuretic peptide caused dose-dependent increases in cGMP production in the purified membrane of colonic tissues. Three types of natriuretic peptide receptor mRNAs were detected using semi-quantitative RT-PCR. Functionally, synthetic DNP inhibited the spontaneous contraction of rat colonic circular muscle in a concentration-dependent manner. The potency appeared to be at least 10 times greater than that of CNP. Furthermore, DNP inhibited carbachol-induced muscle contraction, suggesting that it also can modulate the nerve regulation of colonic motility. This study demonstrates the presence of DNP system in rat colon and its function as a local regulator of colonic motility. [Copyright &y& Elsevier]

Environmental stresses converge on the mitochondria that can trigger or inhibit cell death. Excitable, postmitotic cells, in response to sublethal noxious stress, engage mechanisms that afford protection from subsequent insults. We show that reoxygenation after prolonged hypoxia reduces the reactive oxygen species (ROS) threshold for the mitochondrial permeability transition (MPT) in cardiomyocytes and that cell survival is steeply negatively correlated with the fraction of depolarized mitochondria. Cell protection that exhibits a memory (preconditioning) results from triggered mitochondrial swelling that causes enhanced substrate oxidation and ROS production, leading to redox activation of PKC, which inhibits glycogen synthase kinase-3beta (GSK-3beta). Alternatively, receptor tyrosine kinase or certain G protein-coupled receptor activation elicits cell protection (without mitochondrial swelling or durable memory) by inhibiting GSK-3beta, via protein kinase B/Akt and mTOR/p70(s6k) pathways, PKC pathways, or protein kinase A pathways. The convergence of these pathways via inhibition of GSK-3beta on the end effector, the permeability transition pore complex, to limit MPT induction is the general mechanism of cardiomyocyte protection. [ABSTRACT FROM AUTHOR]

Dendroaspis natriuretic peptide (DNP), a 38-amino acid peptide, was isolated from the venom of green mamba. It has structural and functional similarities to the other members of the natriuretic peptide family. The purpose of this study was to determine whether DNP is present in pig ovarian granulosa cells and to define its biological functions. The serial dilution curves of extracts of granulosa cells and follicular fluid were parallel to the standard curve of DNP, and a major peak of molecular profile of both extracts by HPLC was synthetic DNP. The concentration of DNP was 7.51±1.46 pg/107 cells and 24.81±2.38 pg/ml in granulosa cells and follicular fluid, respectively. Natriuretic peptides increased cGMP production in the purified membrane of granulosa cells with a rank order of potency of C-type natriuretic peptide (CNP)>atrial natriuretic peptide (ANP)=DNP. mRNAs for natriuretic peptide receptor-A (NPR-A), NPR-B and NPR-C were detected by RT-PCR. The binding site of 125I-DNP was also observed in granulosa cell layer by in vitro autoradiography. Synthetic DNP inhibited the secretion of ANP from granulosa cells in a concentration-dependent manner and the potency was similar to CNP. The concentration of DNP and CNP, which inhibited the secretion of ANP by 50%, was about 1 nM. Increases in production of cGMP in granulosa cells were observed by DNP or CNP. Therefore, these results show the existence of DNP system and the cross-talk between natriuretic peptides in pig ovarian granulosa cells. [Copyright &y& Elsevier]

Amylin cosecretes with insulin from pancreatic β-cells and shows high sequence homology with CGRP, adrenomedullin, and salmon calcitonin. This study aimed to investigate the effect of amylin on the atrial hemodynamics and ANP release from rat atria and to identify its receptor subtypes. Isolated perfused left atria from either control or streptozotocin-treated rats were paced at 1.3 Hz. The concentration of ANP was measured by radioimmunoassay and the translocation of ECF was measured by [3H]-inulin clearance. Rat amylin increased atrial contractility and suppressed the release of ANP. Rat CGRP showed similar effects but was approximately 300-fold more potent than amylin. Pretreatment with receptor antagonist for CGRP1 [rat α-CGRP (8-37)] or salmon calcitonin [acetyl-(Asn30, Tyr32)-calcitonin(8-32), (AC 187)] blocked the suppressive effect of ANP release and the positive inotropic effect by rat amylin. However, receptor antagonists for amylin [amylin (8-37), acetyl-amylin] did not block those effects. Amylin (8-37), acetyl-amylin, or rat α-CGRP (8-37) alone accentuated the release of ANP with no changes in atrial contractility. The effect of rat amylin and rat amylin (8-37) on the ANP release was attenuated in streptozotocin-treated rats. We suggest that amylin suppressed ANP release with increased atrial contractility through receptors for CGRP1 and salmon calcitonin and the attenuation of amylin and its antagonist on ANP release from streptozotocin-treated rat atria may be due to the downregulation of amylin receptor. [Copyright &y& Elsevier]

The epithelial amiloride-sensitive sodium channel (ENaC) plays a central role in sodium homeostasis and blood pressure control. The molecular effect of high sodium intake on the ENaC gene is not well known. This study examined the effects of high salt (HS) intake on αENaC gene transcription in rat kidney. Rats were injected intraperitoneally with hypertonic (1.5 M NaCl) or normal saline solution (three rats per group). The serum sodium concentration of rats injected with hypertonic saline increased significantly 30 min after injection (158±2 mM versus 140±1 mM for normal saline injected rats and 139±1 mM for uninjected rats). At 3 h after injection, serum sodium decreased (144±1 mM) but remained above the control values (139±1 mM for normal saline injected rats, 139±1 mM for uninjected rats). The serum aldosterone decreased 1.5 and 3 h after the hypertonic saline injection (217±10 and 139±23 pg/ml for hypertonic saline injected rats, 358±2 pg/ml for uninjected rats). The kidney cortex was dissected macroscopically and total RNA was isolated at 1.5 and 3 h after treatment. Semi-quantitative RT-PCR studies revealed that following hypertonic saline treatment, αENaC mRNA levels were dramatically downregulated, compared with controls, as early as 1.5 h. Western blot analysis showed similar patterns of protein downregulation. Inhibition of protein synthesis by cycloheximide (CHX) blocked the sodium chloride-induced αENaC mRNA downregulation, 3 h after treatment. This indicates that synthesis of new, uncharacterized protein(s) is required for sodium chloride-mediated inhibition of αENaC gene transcription. [Copyright &y& Elsevier]

Calcitonin gene-related peptide (CGRP), a 37-amino acid neuropeptide, is found in the central nervous system as well as in the heart. CGRP shows high sequence homology with amylin, salmon calcitonin, and adrenomedullin. This study aimed to investigate the effect of CGRP on atrial hemodynamics and atrial natriuretic peptide (ANP) release by using isolated perfused beating left atria and to identify its receptor subtypes. Rat α-CGRP (0.1, 1, 10, or 100 nM) increased atrial contractility and suppressed the release of ANP in a concentration-dependent manner. However, cys-CGRP (1 μM), a CGRP2 receptor agonist, slightly decreased ANP release without positive inotropism. Human α-CGRP (1 nM) showed an effect on ANP release similar to that of rat α-CGRP with potent positive inotropism. However, salmon and rat calcitonin (1 μM) caused a slight decrease or no change in ANP release. Pretreatment with a receptor antagonist for CGRP1 [rat α-CGRP-(8–37)] blocked rat α-CGRP-induced suppression of ANP release and positive inotropism, whereas the antagonists for salmon or amylin did not. Therefore, we suggest that rat α-CGRP causes a suppression of ANP release with positive inotropism through the receptor for CGRP1 but not that for calcitonin and amylin. [Copyright &y& Elsevier]

cAMP is known to control the release of atrial natriuretic peptide. To define the roles of cyclic nucleotide phosphodiesterase subtypes in the regulation of atrial natriuretic peptide (ANP) release, experiments were done with perfused beating rabbit atria. Phosphodiesterase 3 subtype-specific inhibitors, milrinone and cilostamide, inhibited myocytic ANP release with a concomitant increase in cAMP efflux. Similarly, trequinsin, another phosphodiesterase 3 inhibitor, decreased ANP release. A phosphodiesterase 4 subtype-specific inhibitor, rolipram, did not significantly change ANP release but increased AMP efflux. Also, 4-[(3-butoxy-4-methoxyphenyl)methyl]-2-imidazolidinone (Ro 20-1724), another phosphodiesterase 4 inhibitor, did not significantly change ANP release. The cAMP efflux was higher in the atrium treated with rolipram than in the atrium treated with milrinone or cilostamide. The data show that the cAMP pool, which is metabolized by phosphodiesterase 3, but not phosphodiesterase 4, is closely related to the basal regulation of atrial ANP release. The results suggest that intracellular cAMP is compartmentalized in the regulation of atrial ANP release, and that the release is controlled by a phosphodiesterase subtype-specific mechanism. [Copyright &y& Elsevier]

• Ang A deteriorated the postischemic heart function via AT1R, which was similar to that by Ang II. • Ang A increased ANP level in coronary effluent and in atrial perfusate but Ang II did not increase it. • Ang A increased blood pressure, which was 10 times less potent than Ang II. • Our results suggest that the first amino acid of Ang II is important to show various physiological functions. Angiotensin (Ang) A differs from Ang II in a single N-terminal alanine residue. The aim of this study was to investigate whether the effects of Ang A on postischemic cardiac injury and hemodynamics differ from Ang II. After euthanizing Sprague–Dawley rats, hearts were perfused with Krebs–Henseleit buffer for a 20 min preischemic period with or without Ang A or Ang II, followed by 20 min global ischemia and 50 min reperfusion. The blood pressure was measured in anesthetized rats. Ang A (0.1, 1.0, 10 μg/kg) deteriorated the postischemic left ventricular hemodynamics in a dose-dependent manner, which was similar to that by Ang II. Ang A (10 μg/kg) increased the infarct size and the lactate dehydrogenase level, and decreased the coronary flow, which were attenuated by the pretreatment with Ang type 1 receptor (AT 1 R) antagonist (losartan) but not by AT 2 R antagonist (PD123319). Ang A increased the expression of apoptotic proteins and decreased the expression of antioxidative proteins. Interestingly, Ang A increased the atrial natriuretic peptide (ANP) level in coronary effluent and in atrial perfusate but Ang II did not increase it. Ang A increased mean arterial blood pressure, which was less potent than Ang II. These results suggest that Ang A has a similar effect on postischemic injury via AT 1 R and less potent vasopressor effect but opposite effect on ANP secretion as compared to Ang II. [ABSTRACT FROM AUTHOR]

• NaHS stimulated ANP secretion during hypoxic condition but not during normoxic condition. • NaHS-induced ANP secretion was attenuated by KATP channel inhibitor but enhanced by KATP channel opener. • The augmentation of NaHS-induced ANP secretion during hypoxia was attenuated by HIF-1α and PPAR-γ inhibitor. • NaHS stimulated ANP secretion during hypoxic condition via KATP channel and HIF-1α and PPAR-γ pathway. It has been reported that sodium hydrosulfide (NaHS) stimulated high stretch induced-atrial natriuretic peptide (ANP) secretion via ATP sensitive potassium (K ATP) channel. K ATP channel is activated during hypoxic condition as a compensatory mechanism. However, whether NaHS affects ANP secretion during hypoxia remains obscure. The purpose of the present study is to discover the impact of NaHS on ANP secretion during hypoxia and to unravel its signaling pathway. Isolated beating rat atria were perfused with buffer exposed to different O 2 tension (to 100% O 2 , normoxia; to 20% O 2 , hypoxia). The ANP secretion increased negatively correlated with O 2 tension. NaHS (50 μM) did not show any significant effect on low stretch induced-ANP secretion in normoxic condition but augmented low stretch induced-ANP secretion in hypoxic condition. The augmentation of NaHS-induced ANP secretion during hypoxia was blocked by the pretreatment with K ATP channel blocker (glibenclamide) and was enhanced by the pretreatment with K ATP channel activator (pinacidil). Hypoxia increased the expression of PPAR-γ protein but did not change the expression of HIF-1α protein and eNOS phosphorylation. The NaHS-induced ANP secretion during hypoxia was also blocked by the pretreatment with HIF-1α inhibitor (2-methoxy- estradiol), PPAR−γ inhibitor (GW9662) but not by NOS inhibitor (L-NAME) and endothelin receptor inhibitor (bosentan). The intravenous infusion of NaHS increased plasma ANP level in monocrotaline-treated rats but not in sham rats. These results suggest that hypoxia augmented NaHS-induced ANP secretion partly through K ATP channel, HIF-1α, and PPAR−γ pathway. [ABSTRACT FROM AUTHOR]

Purpose: Renin-angiotensin system (RAS) is involved in the pathophysiology of colonic inflammation. The aim of this study was to investigate whether small angiotensins (Angs) peptides play a role in the regulation of colonic motility and their roles are modulated in colitis., Methods: Experimental colitis was induced by an intake of 5% dextran sulfate sodium (DSS) dissolved in tap water for 7 days in Sprague-Dawley rats. After sacrifice, plasma hormone concentrations and messenger RNAs (mRNAs) for RAS were measured. Functional analysis of colonic motility in response to Angs peptides was performed using Taenia coli., Results: DSS-treated colon showed an increased necrosis with massive infiltration of inflammatory cells. The mRNA level of colonic angiotensin II receptor type 2 (AT2R) in DSS-treated rats was higher than that in control rats whereas the mRNA levels of angiotensin II converting enzyme (ACE), ACE2, AT1R, AT4R, and Mars receptor were not different from those in control rats. Ang III, Ang IV, and Ang-(1-9) (1, 3 μM) increased the frequency of basal colonic motility. Ang-(1-7) did not cause any significant changes in frequency and amplitude of basal motility. The order of potency for an increased frequency of basal motility seems to be Ang II>>Ang IV>Ang III=Ang-(1-9). The increased frequency of basal motility by Ang-(1-9) but not Ang IV was significantly enhanced in DSS-treated rat colon., Conclusion: In conclusion, these data suggest that small Angs peptides are partly involved in the pathophysiological regulation of colonic motility in experimental colitis.

The lack of a clonal renin-secreting cell line has greatly hindered the investigation of the regulatory mechanisms of renin secretion at the cellular, biochemical, and molecular levels. In the present study, we investigated whether it was possible to induce phenotypic switching of the renin-expressing clonal cell line As4.1 from constitutive inactive renin secretion to regulated active renin secretion. When grown to postconfluence for at least two days in media containing fetal bovine serum or insulin-like growth factor-1, the formation of cell-cell contacts via N-cadherin triggered downstream cellular signaling cascades and activated smooth muscle-specific genes, culminating in phenotypic switching to a regulated active renin secretion phenotype, including responding to the key stimuli of active renin secretion. With the use of phenotype-switched As4.1 cells, we provide the first evidence that active renin secretion via exocytosis is regulated by phosphorylation/dephosphorylation of the 20 kDa myosin light chain. The molecular mechanism of phenotypic switching in As4.1 cells described here could serve as a working model for full phenotypic modulation of other secretory cell lines with incomplete phenotypes.

Carbon monoxide (CO) is a known gaseous bioactive substance found across a wide array of body systems. The administration of low concentrations of CO has been found to exert an anti-inflammatory, anti-apoptotic, anti-hypertensive, and vaso-dilatory effect. To date, however, it has remained unknown whether CO influences atrial natriuretic peptide (ANP) secretion. This study explores the effect of CO on ANP secretion and its associated signaling pathway using isolated beating rat atria. Atrial perfusate was collected for 10 min for use as a control, after which high atrial stretch was induced by increasing the height of the outflow catheter. Carbon monoxide releasing molecule-2 (CORM-2; 10, 50, 100 μM) and hemin (HO-1 inducer; 0.1, 1, 50 μM), but not CORM-3 (10, 50, 100 μM), decreased high stretch-induced ANP secretion. However, zinc porphyrin (HO-1 inhibitor) did not affect ANP secretion. The order of potency for the suppression of ANP secretion was found to be hemin > CORM-2 >> CORM-3. The suppression of ANP secretion by CORM-2 was attenuated by pretreatment with 5-hydroxydecanoic acid, paxilline, and 1 H -[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one, but not by diltiazem, wortmannin, LY-294002, or NG-nitro-L-arginine methyl ester. Hypoxic conditions attenuated the suppressive effect of CORM-2 on ANP secretion. In sum, these results suggest that CORM-2 suppresses ANP secretion via mitochondrial KATP channels and large conductance Ca 2+ -activated K + channels.

Background: Alamandine differs from angiotensin-(1-7) in a single N-terminal alanine residue. The aim of this study was to investigate whether alamandine protects the heart against reperfusion injury., Methods and results: After euthanizing Sprague-Dawley rats, hearts were perfused with Krebs-Henseleit buffer for a 20-min pre-ischemic period with or without alamandine, followed by 20 min global ischemia and 50 min reperfusion. Alamandine (0.1 mg/kg) improved the postischemic left ventricular developed pressure and ±dP/dt, decreased the infarct size, and decreased the lactate dehydrogenase levels in the effluent. Alamandine increased the coronary flow and the amount of atrial natriuretic peptide (ANP) in the coronary effluent, and it decreased the expression of apoptotic proteins and increased the expression of antioxidative proteins. Pretreatment with the MrgD receptor antagonist or PD123319, but not the angiotensin type 1 receptor antagonist, attenuated the cardioprotective effects of alamandine. A similar cardioprotective effect with alamandine was also observed with high plasma ANP levels in an in vivo study. Alamandine directly stimulated ANP secretion from isolated atria, which was completely blocked by pretreatment with the MrgD receptor antagonist and was partially blocked by PD123319., Conclusions: These results suggest that the cardioprotective effects of alamandine against I/R injury are, in part, related to the activation of antioxidant and antiapoptotic enzymes via the MrgD receptor.

Suberoyl bishydroxamic acid (SBHA) as a histone deacetylase (HDAC) inhibitor can induce apoptosis through the formation of reactive oxygen species (ROS). However, there is no report about the regulation of ROS and antioxidant enzymes in SBHA-treated lung cancer cells. Here, we investigated the toxicological effects of SBHA on the regulations of ROS, glutathione (GSH), and antioxidant enzymes, especially thioredoxin (Trx) in A549 lung cancer cells. SBHA inhibited the growth of A549 cells in time- and dose-dependent manners, and it induced apoptosis which accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨm). SBHA significantly increased ROS levels including O2 (•-) level at 72 h whereas it decreased ROS levels at the early time points (30 min to 3 h). SBHA also induced GSH depletion at 24 and 72 h. N-acetyl cysteine (NAC; a well-known antioxidant) prevented apoptotic cell death and GSH depletion via decreasing ROS in SBHA-treated A549 cells. In addition, SBHA changed the levels of antioxidant-related proteins, especially Trx1. The expression and activity of Trx1 in A549 cells were reduced by SBHA. While the downregulation of Trx1 enhanced cell death, ROS level, and GSH depletion in SBHA-treated A549 cells, the overexpression of Trx1 decreased ROS level in these cells without the prevention of cell death and GSH depletion. In conclusion, SBHA-induced A549 cell death was influenced by changes in ROS and GSH levels. The basal status of Trx1 among other antioxidant proteins was closely correlated with the survival of A549 cells.

Auranofin (Au), an inhibitor of thioredoxin reductase, is a known anti‑cancer drug. In the present study, the anti‑growth effect of Au on HeLa cervical cancer cells was examined in association with levels of reactive oxygen species (ROS) and glutathione (GSH). Au inhibited the growth of HeLa cells with an IC50 of ~2 µM at 24 h. This agent induced apoptosis and necrosis, accompanied by the cleavage of poly (ADP‑ribose) polymerase and loss of mitochondrial membrane potential. The pan‑caspase inhibitor, benzyloxycarbonyl‑Val‑Ala‑Asp‑fluoromethylketone, prevented apoptotic cell death and each of the assessed caspase inhibitors inhibited necrotic cell death induced by Au. With respect to the levels of ROS and GSH, Au increased intracellular O2•- in the HeLa cells and induced GSH depletion. The pan‑caspase inhibitor reduced the levels of O2•- and GSH depletion in Au‑treated HeLa cells. The antioxidant, N‑acetyl cysteine, not only attenuated apoptosis and necrosis in the Au‑treated HeLa cells, but also decreased the levels of O2•- and GSH depletion in the cells. By contrast, L‑buthionine sulfoximine, a GSH synthesis inhibitor, intensified cell death O2•- and GSH depletion in the Au‑treated HeLa cells. In conclusion, Au induced apoptosis and necrosis in HeLa cells via the induction of oxidative stress and the depletion of GSH.

Our previous study demonstrated that the concentration of dendroaspis natriuretic peptide (DNP) was markedly higher than that of atrial NP (ANP) in rabbit plasma, indicating that DNP has a different metabolic rate from other NPs. Therefore, the metabolic characteristics of DNP in mammals require further analysis. The stabilities of NPs were determined by incubating 125I‑labeled ANP, brain NP (BNP), C‑type NP (CNP) and DNP at 37˚C for 1, 2 and 4 h, and analyzing their profiles by reversed‑phase high‑performance liquid chromatography. 125I‑labeled ANP, BNP and CNP were quickly degraded in rat plasma, while 125I‑labeled DNP was stable for 4 h. The relative stability of the peptides following incubation in rat plasma followed the rank order of: DNP>>>ANP≥BNP>>CNP. Organs were also examined for the degradation of DNP, including the spleen, kidney, liver, heart and lung. The physiological target organ for the degradation of DNP was observed to be the kidney. Furthermore, degradation of DNP in the kidney was attenuated by phenanthroline, a metalloproteinase inhibitor. Therefore, these results indicate that DNP has a longer stability in plasma and that it may have strong therapeutic applications in cardiac disease.

Angiotensin-(1-7) [ANG-(1-7)] plays a counterregulatory role to angiotensin II in the renin-angiotensin system. In trained spontaneous hypertensive rats, Mas expression and protein are upregulated in ventricular tissue. Therefore, we examined the role of ANG-(1-7) on cardiac hemodynamics, cardiac functions, and cardiac remodeling in trained two-kidney one-clip hypertensive (2K1C) rats. For this purpose, rats were divided into sedentary and trained groups. Each group consists of sham and 2K1C rats with and without ANG-(1-7) infusion. Swimming training was performed for 1 h/day, 5 days/wk for 4 wk following 1 wk of swimming training for acclimatization. 2K1C rats showed moderate hypertension and left ventricular hypertrophy without changing left ventricular function. Chronic infusion of ANG-(1-7) attenuated hypertension and cardiac hypertrophy only in trained 2K1C rats but not in sedentary 2K1C rats. Chronic ANG-(1-7) treatment significantly attenuated increases in myocyte diameter and cardiac fibrosis induced by hypertension in only trained 2K1C rats. The Mas receptor, ANG II type 2 receptor protein, and endothelial nitric oxide synthase phosphorylation in ventricles were upregulated in trained 2K1C rats. In conclusion, chronic infusion of ANG-(1-7) attenuates hypertension in trained 2K1C rats.

Arsenic trioxide (ATO; As(2)O(3)) can induce apoptotic cell death in various cancer cells including lung cancer cells. However, little is known about the toxicological effects of ATO on normal primary lung cells. In this study, we investigated the cellular effects of ATO on human pulmonary fibroblast (HPF) cells in relation to cell growth inhibition and death. ATO inhibited HPF cell growth with an IC(50) of approximately 30-40 μM at 24 h and induced cell death accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ(m)). Thus, HPF cells were considered to be very resistant to ATO insults. ATO increased the expression of p53 protein and decreased that of Bcl-2 protein. This agent activated caspase-8 but not caspase-3 in HPF cells. Z-VAD (a pan-caspase inhibitor; 15 μM) did not significantly decrease cell growth inhibition, death and MMP (ΔΨ(m)) loss by ATO. Moreover, administration of Bax or casase-8 siRNA attenuated HPF cell death by ATO whereas p53 or caspase-3 siRNAs did not affect cell death. In conclusion, HPF cells were resistant to ATO and higher doses of ATO induced the growth inhibition and death in HPF cells via the regulation of Bcl-2 family and caspase-8.

Reactive oxygen species (ROS) and Ca(2+) signals are closely associated with the pathogenesis of cardiac hypertrophy. However, the cause and effect of the two signals in cardiac hypertrophy remain to be clarified. We extend our recent report by investigating a potential interaction between ROS and Ca(2+) signals utilizing in vitro and in vivo angiotensin II (ANG II)-induced cardiac hypertrophy models. ANG II-induced initial Ca(2+) transients mediated by inositol trisphosphate (IP(3)) triggered initial ROS production in adult rat cardiomyocytes. The ROS generated by activation of the NAD(P)H oxidase complex via Rac1 in concert with Ca(2+) activates ADP-ribosyl cyclase to generate cyclic ADP-ribose (cADPR). This messenger-mediated Ca(2+) signal further augments ROS production, since 2,2'-dihydroxyazobenzene, an ADP-ribosyl cyclase inhibitor, or 8-Br-cADPR, an antagonistic analog of cADPR, abolished further ROS production. Data from short hairpin RNA (shRNA)-mediated knockdown of Akt1 and p47(phox) demonstrated that Akt1 is the upstream key molecule responsible for the initiation of Ca(2+) signal that activates p47(phox) to generate ROS in cardiomyocytes. Nuclear translocation of nuclear factor of activated T-cell in cardiomyocytes was significantly suppressed by treatment with NAD(P)H oxidase inhibitors as well as by shRNA against Akt1 and p47(phox). Our results suggest that in cardiomyocytes Ca(2+) and ROS messengers generated by ANG II amplify the initial signals in a cooperative manner, thereby leading to cardiac hypertrophy.

Paclitaxel (taxol) has been used for the treatment of various human tumors and is an exceedingly efficient chemotherapy agent against esophageal cancer. However, the precise molecular mechanisms of paclitaxel effects on human esophageal adenocarcinoma cells are not well understood. MTT assay and cell cycle analysis were performed to examine the mechanism of antiproliferative and cell viability effects of paclitaxel in human esophageal adenocarcinoma cancer cells. Western blotting was also used to examine the cell cycle- and apoptosis-related proteins. Paclitaxel inhibited the proliferation of SKGT4 cells in a dose- and time-dependent manner with G2/M arrest. In addition, paclitaxel induced apoptosis through the activation of caspase-3 followed by PARP degradation. In conclusion, our results suggest that paclitaxel leads to mitotic cell cycle arrest following G2/M arrest and induces apoptosis via a caspase-3 pathway in SKGT4 cells.

Gallic acid (GA) is generally distributed in a variety of plants and foods, and its various biological effects have been reported. Here, we investigated the effects of GA and/or caspase inhibitors on Calu-6 and A549 lung cancer cells in relation to cell death and reactive oxygen species (ROS). The growths of Calu-6 and A549 cells were diminished with an IC(50) of approximately 30 and 150 μM GA at 24 h, respectively. GA also inhibited the growth of primary human pulmonary fibroblast (HPF) cells with an IC(50) of about 300 μM. GA induced apoptosis and/or necrosis in lung cancer cells, which was accompanied by the loss of mitochondrial membrane potential (MMP, ΔΨ(m)). The percents of MMP (ΔΨ(m)) loss and death cells by GA were lower in A549 cells than in Calu-6 cells. Caspase inhibitors did not significantly rescued lung cancer cells from GA-induced cell death. GA increased ROS levels including O(2) (•-) and induced GSH depletion in both lung cancer cells. Z-VAD (pan-caspase inhibitor) did not decrease ROS levels and GSH depleted cell number in GA-treated lung cancer cells. In conclusion, GA inhibited the growth of lung cancer and normal cells. GA-induced lung cancer cell death was accompanied by ROS increase and GSH depletion.

Objective: • To evaluate the impact and distribution of a single phosphodiesterase type 5 inhibitor (PDE5 I) dose (udenafil or tadalafil) in prostate tissue and plasma in patients with benign prostatic hyperplasia (BPH)., Patients and Methods: • Thirty BPH patients complaining of erectile dysfunction along with moderate-to-severe lower urinary tract symptoms (LUTS) who underwent transurethral resection of the prostate (TURP) were enrolled in the present study. • The patients were randomly divided into the three groups: group 1, TURP without PDE5 Is; group 2, 200 mg of udenafil given 1 h before TURP; and group 3, 20 mg of tadalafil given 1 h before TURP. • We evaluated the concentrations of PDE5-I, cAMP and cGMP in prostate tissues and plasma, and calculated the prostate tissue-to-plasma (T/P) ratio., Results: • The concentration of udenafil in prostate tissue and plasma was 2028.6 ± 360.8 ng/g and 463.7 ± 39.1 ng/mL, respectively, and the resulting T/P ratio was 4.4. The tadalafil concentration in prostate tissue and plasma was 385.7 ± 83.8 ng/g and 305.8 ± 41.1 ng/mL, respectively, and the T/P ratio was 1.3. • Udenafil and tadalafil significantly increased the cAMP and cGMP levels in plasma and prostate tissues., Conclusions: • Udenafil and tadalafil significantly increased cAMP and cGMP levels and were more highly distributed in the prostate than plasma. The T/P ratio of udenafil was higher than tadalafil. • These findings may help in the assessment of the feasibility of using PDE5 Is to concurrently treat both LUTS and erectile dysfunction., (© 2010 THE AUTHORS. BJU INTERNATIONAL © 2010 BJU INTERNATIONAL.)

Urotensin II (UII) is a vasoactive peptide with potent cardiovascular effects through a G protein-coupled receptor. Hypoxia stimulates the secretion of UII and atrial natriuretic peptide (ANP). However, the effect of UII on hypoxia-induced cardiac hypertrophy is still controversial. The present study was conducted to determine whether human UII (hUII)-mediated ANP secretion influences hypoxia-induced cardiac hypertrophy using in vitro and in vivo models. Hypoxia caused an increase in ANP secretion and a decrease in atrial contractility in isolated perfused beating rat atria. hUII (0.01 and 0.1 nM) attenuated hypoxia-induced ANP secretion without changing the atrial contractility, and the hUII effect was mediated by the UII receptor signaling involving phospholipase C, inositol 1,3,4 trisphosphate receptor, and protein kinase C. Rats treated with monocrotaline (MCT, 60 mg/kg) showed right ventricular hypertrophy with increases in pulmonary arterial pressure and its diameter and plasma levels of UII and ANP that were attenuated by the pretreatment with an UII receptor antagonist, urantide. An acute administration of hUII (5 μM injection plus 2.5 μM infusion for 15 min) decreased the plasma ANP level in MCT-treated rats but increased the plasma ANP level in MCT plus urantide-treated and sham-operated rats. These results suggest that hUII may deteriorate MCT-induced cardiac hypertrophy mainly through a vasoconstriction of the pulmonary artery and partly through the suppression of ANP secretion.

Propyl gallate (PG) as a synthetic antioxidant exerting a variety of effects on tissue and cell functions. We evaluated the effects of PG on the growth of endothelial cells, especially calf pulmonary artery endothelial cells (CPAEC) in relation to apoptosis. PG dose-dependently inhibited the growth of CPAEC and human umbilical vein endothelial cells (HUVEC) at 24 h. The susceptibility of CPAEC to PG was higher than that of HUVEC. PG induced apoptosis in CPAEC, which was accompanied by the loss of mitochondrial membrane potential (MMP; DeltaPsim). The tested caspase inhibitors (pan-caspase, caspase-3, -8 or -9 inhibitor) did not rescue CPAEC from PG-induced cell death but instead slightly enhanced the cell death. PG increased reactive oxygen species (ROS) level in CPAEC. The caspase inhibitors did not significantly change the ROS level. Furthermore, PG increased the GSH depleted cell number and decreased GSH level in CPAEC. The tested caspase inhibitors did not significantly change the number in PG-treated CPAEC. Each caspase inhibitor differently alters GSH levels in CPAEC. In conclusion, PG inhibited the growth of endothelial cells, especially CPAEC via caspase-independent apoptosis. PG-induced CPAEC death was accompanied by ROS increase and GSH depletion.

Gallic acid (GA) is widely distributed in various plants and foods and has various biological effects. In this study, we investigated the effects of mitogen-activated protein kinase (MEK, JNK or p38) inhibitors on GA-induced Calu-6 lung cancer cell death in relation to reactive oxygen species (ROS) and glutathione (GSH) levels. GA inhibited the growth of Calu-6 cells and induced apoptosis and/or necrosis accompanied by the loss of mitochondrial membrane potential (MMP; Δψm). ROS levels and the number of GSH-depleted cells were observed to be increased at 24 h. MEK inhibitor suppressed cell growth inhibition, death, MMP (Δψm) loss and GSH depletion induced by GA, but failed to suppress the increase in ROS levels. JNK inhibitor also somewhat suppressed cell growth inhibition, MMP (Δψm) loss and GSH depletion induced by GA, and limited the increase in ROS levels. By contrast, p38 inhibitor mildly enhanced GA-induced cell growth inhibition, MMP (Δψm) loss and the increase in ROS levels. In conclusion, MEK inhibitor suppressed GA-induced cell growth inhibition and death in Calu-6 cells. This was related to the prevention of GSH depletion.

Angiotensin-(1-7) [ANG-(1-7)], one of the bioactive peptides produced in the renin-angiotensin system, plays a pivotal role in cardiovascular physiology by providing a counterbalance to the function of ANG II. Recently, it has been considered as a potential candidate for therapeutic use in the treatment of various types of cardiovascular diseases. The aim of the present study is to explain the modulatory role of ANG-(1-7) in atrial natriuretic peptide (ANP) secretion and investigate the functional relationship between two peptides to induce cardiovascular effects using isolated perfused beating rat atria and a cardiac hypertrophied rat model. ANG-(1-7) (0.01, 0.1, and 1 muM) increased ANP secretion and ANP concentration in a dose-dependent manner at high atrial pacing (6.0 Hz) with increased cGMP production. However, at low atrial pacing (1.2 Hz), ANG-(1-7) did not cause changes in atrial parameters. Pretreatment with an antagonist of the Mas receptor or with inhibitors of phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), or nitric oxide synthase blocked the augmentation of high atrial pacing-induced ANP secretion by ANG-(1-7). A similar result was observed with the inhibition of the Na(+)/H(+) exchanger-1 and Ca(2+)/calmodulin-dependent kinase II (CaMKII). ANG-(1-7) did not show basal intracellular Ca(2+) signaling in quiescent atrial myocytes. In an in vivo study using an isoproterenol-induced cardiac hypertrophy animal model, an acute infusion of ANG-(1-7) increased the plasma concentration of ANP by twofold without changes in blood pressure and heart rate. A chronic administration of ANG-(1-7) increased the plasma ANP level and attenuated isoproterenol-induced cardiac hypertrophy. The antihypertrophic effect was abrogated by a cotreatment with the natriuretic peptide receptor-A antagonist. These results suggest that 1) ANG-(1-7) increased ANP secretion at high atrial pacing via the Mas/PI3K/Akt pathway and the activation of Na(+)/H(+) exchanger-1 and CaMKII and 2) ANG-(1-7) decreased cardiac hypertrophy which might be mediated by ANP.

Introduction: The present study introduces a newly devised in vitro penile perfusion model, which allows the measurement of changes in intracavernosum pressure (ICP), tension, and cyclic nucleotides in penile tissue in a one-step procedure., Aims: We compared the penile perfusion model with the classical penile strip chamber model to develop the new model., Methods: We investigated the role of nitric oxide-cyclic guanosine monophosphate pathway in both systems activated by acetylcholine or electrical field stimulation (EFS). We measured cGMP (cyclic guanosine monophosphate) in the penile corpus cavernosum smooth muscle in both systems and perfusates in the penile perfusion model. We determined ICP, tension, and cGMP simultaneously in penile perfusion model during the experiments., Main Outcome Measures: Changes of tension and cGMP in both models, and of cGMP in perfusates and of ICP in penile perfusion model., Results: In both systems, acetylcholine relaxed the phenylephrine-precontracted penile corpus cavernosum smooth muscle in a concentration-dependent manner. EFS decreased the tension in both models and ICP in the penile perfusion model in a frequency-dependent manner. Pretreatment with atropine, Nomega nitro-L-arginine-methyl ester (L-NAME), and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) blocked the acetylcholine-induced decrease in the tension in both models and ICP in penile perfusion model. Acetylcholine increased cGMP levels in the perfusate of penile perfusion model and the changes were attenuated by tetrodotoxin (TTX), atropine, L-NAME and ODQ. EFS increased cGMP levels of the penile corpus cavernosum smooth muscle of both models and the changes were attenuated by TTX, atropine, L-NAME, and ODQ., Conclusion: The results indicated that the responses of penile strip and perfused penile tissue to acetylcholine and EFS were very similar in both models in terms of tension and cGMP levels. Furthermore, the new model has more advantages over the classical penile strip chamber model in measuring intracavernosum metabolic changes and ICP without interruption of the procedure.

MG132 as a proteasome inhibitor has been shown to induce apoptotic cell death through formation of reactive oxygen species (ROS). Here, we investigated the effects of N-acetyl cysteine (NAC; a well-known antioxidant), L-buthionine sulfoximine (BSO; an inhibitor of GSH synthesis) or diethyldithiocarbamate (DDC; an inhibitor of Cu/Zn-SOD) on MG132-treated Calu-6 or A549 lung cancer cells in relation to cell growth, ROS and GSH levels. MG132 inhibited the growth of Calu-6 and A549 cells at 24 h. MG132 induced apoptosis in both cell lines, which was accompanied by the loss of mitochondrial membrane potential (MMP; DeltaPsim). ROS levels including O(2)(.-) were increased in both MG132-treated lung cells. MG132 also induced GSH depletion in both lung cell types. Treatment with 10 microM BSO or 1 microM DDC affected ROS and GSH levels in MG132-treated Calu-6 cells. However, these changes did not influence cell growth and death in the cells. NAC prevented cell growth inhibition and death in MG132-treated lung cells, which was accompanied by decreased ROS, but not by decreased GSH depletion. In conclusion, the changes of ROS and GSH by MG132, NAC, BSO or DDC were partially related to cell growth and death in the lung cancer cell lines Calu-6 and A549.

Leptin is a circulating adipocyte-derived hormone that influences blood pressure (BP) and metabolism. This study was designed to define the possible role of leptin in regulation of the atrial natriuretic peptide (ANP) system using acute and chronic experiments. Intravenous infusion of rat leptin (250 microg/kg injection plus 2 microg.kg(-1).min(-1) for 20 min) into Sprague-Dawley rats increased BP by 25 mmHg and decreased plasma level of ANP from 80.3 +/- 3.45 to 51.8 +/- 3.3 pg/ml. Reserpinization attenuated the rise in BP, but not the reduction of plasma ANP during leptin infusion. N(omega)-nitro-l-arginine methyl ester prevented the effects of leptin on the reduction of ANP level. In hyperleptinemic rats that received adenovirus containing rat leptin cDNA (AdCMV-leptin), BP increased during first 2 days and then recovered to control value. Plasma concentration of ANP and expression of ANP mRNA, but not of atrial ANP, in hyperleptinemic rats were lower than in the control groups on the first and second week after administration of AdCMV-leptin. These effects were not observed by the pretreatment with N(omega)-nitro-l-arginine methyl ester. No differences in renal function and ANP receptor density in the kidney were found between hyperleptinemic and control rats. Basal ANP secretion and isoproterenol-induced suppression of ANP secretion from isolated, perfused atria of hyperleptinemic rats were not different from those of other control groups. These data suggest that leptin inhibits ANP secretion indirectly through nitric oxide without changing basal or isoproterenol-induced ANP secretion.

Propyl gallate (PG) as a synthetic antioxidant exerts a variety of effects on tissue and cell functions. Here, we evaluated the effects of PG on the growth of HeLa cells in relation to apoptosis and the cell cycle. PG dose-dependently inhibited the growth of HeLa cells with an IC50 of approximately 800 microM at 24 h. DNA flow cytometric analysis indicated that PG significantly induced a G1 phase arrest of the cell cycle along with an increase in the cyclin-dependent kinase inhibitor (CDKI) p27. In addition, PG induced apoptosis, which was accompanied by the loss of mitochondrial membrane potential (MMP; DeltaPsim), activation of caspase-3 and caspase-8 and PARP cleavage. All the tested caspase inhibitors (pan-caspase, caspase-3, -8 or -9 inhibitor) significantly rescued HeLa cells from PG-induced cell death. However, none of the caspase inhibitors prevented the loss of MMP (DeltaPsim) induced by PG. In conclusion, PG inhibited the growth of HeLa cells via caspase-dependent apoptosis as well as a G1 phase arrest of the cell cycle.

MG132, a proteasome inhibitor, has been shown to induce apoptotic cell death through formation of reactive oxygen species (ROS). Here, we evaluated the effects of MG132 on the growth of endothelial cells, especially calf pulmonary artery endothelial cells (CPAECs). MG132 dose-dependently inhibited the growth of CPAECSs and human umbilical vein endothelial cells (HUVECs) at 24 hours. MG132 also induced apoptosis in both cell lines, which was accompanied by the loss of mitochondrial membrane potential. All the tested caspase inhibitors (pan-caspase, caspase-3, -8 and -9 inhibitor) significantly rescued CPAECs from MG132-induced cell death. MG132 increased ROS level and GSH depleted cell numbers of CPAECs. None of the caspase inhibitors reduced ROS level in MG132-treated CPAECs but did reduce apoptosis in these cells. In conclusion, MG132 inhibited the growth of endothelial cells, especially CPAECs via caspase-dependent apoptosis. MG132-induced CPAEC death was related to GSH depletion rather than a change in ROS level.

Arsenic trioxide (ATO) can regulate many biological functions such as apoptosis and differentiation. We evaluated the effects of ATO on various cell types such as cervical cancer HeLa cells, pulmonary adenocarcinoma Calu-6 and A549 cells, calf pulmonary artery endothelial cells (CPAEC), human umbilical vein endothelial cells (HUVEC) and human pulmonary fibroblast (HPF) cells in relation to cell growth, cell death and reactive oxygen species (ROS) and glutathione (GSH) levels. The growth of HeLa and Calu-6 cells was inhibited by ATO with an IC50 of approximately 15 microM at 24 h. A549 cell growth was not inhibited by 15 microM ATO. The susceptibility to ATO in CPAEC and HUVEC was similar to that in HeLa cells. The IC50 of ATO in HPF cells was approximately 40 microM. ATO induced apoptosis in HeLa, CPAEC and HUVEC, which was accompanied by the loss of mitochondrial membrane potential (DeltaPsim). However, ATO did not strongly trigger apoptosis in Calu-6, A549 and HPF cells. ATO increased or decreased the ROS level including O2.- and GSH levels depending on the incubation dose and cell type. In conclusion, ATO differentially affected cell growth inhibition and death depending on the incubation dose and cell type. The changes in ROS and GSH levels by ATO were not tightly correlated with the level of cell death. Our present data provide useful information for the action of ATO in various cell types in relation to cell growth, cell death, ROS and GSH levels.

Pyrogallol (PG) as a polyphenol compound induces apoptosis in several types of cells. Here, we evaluated the effects of PG on endothelial cells (ECs), especially calf pulmonary artery endothelial cells (CPAEC) in relation to the cell growth, ROS and glutathione (GSH) levels. PG dose-dependently inhibited the growth of CPAEC and human umbilical vein endothelial cells (HUVEC) at 24 h. PG also induced apoptosis in CPAEC, which was accompanied by the loss of mitochondrial membrane potential (MMP; DeltaPsim). PG decreased ROS level including O2*- and PG dose-dependently increased GSH depleted cell number in both EC types. N-acetyl-cysteine (NAC; a well-known antioxidant) increased ROS levels in PG-treated CPAEC with the prevention of cell death and GSH depletion. In conclusion, PG inhibited the growth of ECs, especially CPAEC via apoptosis. PG-induced EC death was related to GSH depletion rather than ROS level changes.