Search

Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) is an uncoupler of mitochondrial oxidative phosphorylation in eukaryotic cells. Here, we investigated an involvement of O(2)(*-) and GSH in FCCP-induced Calu-6 cell death and examined whether ROS scavengers rescue cells from FCCP-induced cell death. Levels of intracellular O(2)(*-) were markedly increased depending on the concentrations (5-100 microM) of FCCP. A depletion of intracellular GSH content was also observed after exposing cells to FCCP. Stable SOD mimetics, Tempol and Tiron did not change the levels of intracellular O(2)(*-), apoptosis and the loss of mitochondrial membrane potential (DeltaPsi(m)). Treatment with thiol antioxidants, NAC and DTT, showed the recovery of GSH depletion and the reduction of O(2)(*-) levels in FCCP-treated cells, which were accompanied by the inhibition of apoptosis. In contrast, BSO, a well-known inhibitor of GSH synthesis, aggravated GSH depletion, oxidative stress of O(2)(*-) and cell death in FCCP-treated cells. Taken together, our data suggested that FCCP as an O(2)(*-) generator, induces apoptosis via the depletion of intracellular GSH contents in Calu-6 cells.

2,4-Dinitrophenol (DNP) is an uncoupler of oxidative phosphorylation in mitochondria. Here, we investigated the in vitro effect of DNP on apoptosis and the involvement of reactive oxygen species (ROS) in As4.1 juxtaglomerular cell death. Dose- and time-dependent induction of apoptosis was evidenced by flow cytometric detection of sub-G1 DNA content and annexin V binding assay. The intracellular H(2)O(2) and O(2)(-) levels were markedly increased in DNP-treated cells. However, the reduction of intracellular H(2)O(2) level by Tiron and catalase did not prevent apoptosis induced by DNP. Moreover, DNP rapidly reduced intracellular GSH content in As4.1 cells. Taken together, apoptosis in DNP-treated As4.1 cells is correlated with the rapid change of intracellular GSH levels rather than ROS levels.

Neuropeptide Y (NPY) receptors are present in cardiac membranes. However, its physiological roles in the heart are not clear. The aim of this study was to define the direct effects of pancreatic polypeptide (PP) on atrial dynamics and atrial natriuretic peptide (ANP) release in perfused beating atria. Pancreatic polypeptides, a NPY Y(4) receptor agonist, decreased atrial contractility but was not dose-dependent. The ANP release was stimulated by PP in a dose-dependent manner. GR 23118, a NPY Y(4) receptor agonist, also increased the ANP release and the potency was greater than PP. In contrast, peptide YY (3-36) (PYY), an NPY Y(2) receptor agonist, suppressed the release of ANP with positive inotropy. NPY, an agonist for Y(1, 2, 5) receptor, did not cause any significant changes. The pretreatment of NPY (18-36), an antagonist for NPY Y(3) receptor, markedly attenuated the stimulation of ANP release by PP but did not affect the suppression of ANP release by PYY. BIIE0246, an antagonist for NPY Y(2) receptor, attenuated the suppression of ANP release by PYY. The responsiveness of atrial contractility to PP or PYY was not affected by either of the antagonists. These results suggest that NPY Y(4) and Y(2) receptor differently regulate the release of atrial ANP.

Pyrogallol (PG) is a polyphenol compound and is known to be an O2.- generator. In the present study, we evaluated the anti-apoptotic effects of caspase inhibitors in relation to changes in reactive oxygen species (ROS) and glutathione (GSH) levels in PG-treated human pulmonary adenocarcinoma Calu-6 cells. Treatment with 50 microM PG inhibited the growth of Calu-6 cells approximately 60% and induced apoptosis approximately 17% at 24 h, accompanied by mitochondrial membrane potential loss (DeltaPsim). Treatment with pan-caspase inhibitor (Z-VAD-FMK), caspase-3 inhibitor (Z-DEVD-FMK), caspase-8 inhibitor (Z-IETD-FMK) and caspase-9 inhibitor (Z-LEHD-FMK) significantly prevented apoptosis in PG-treated Calu-6 cells at 24 h. PG increased the ROS and depleted GSH contents in Calu-6 cells. Treatment with each caspase inhibitor did not significantly change the ROS and GSH levels in PG-treated Calu-6 cells at 24 h. However, Z-VAD significantly prevented GSH depletion in PG-treated Calu-6 cells at the late time phase of 72 h. Conclusively, the anti-apoptotic effect of caspase inhibitor on PG-induced Calu-6 cell death was closely related to changes in GSH content rather than ROS levels.

Antimycin A (AMA) inhibits mitochondrial electron transport between cytochromes b and c. We evaluated the effects of AMA on the growth of human pulmonary adenocarcinoma A549 cells in relation to cell cycle and apoptosis. Treatment with 2-100 microM AMA significantly inhibited the cell growth of A549 for 72 h. DNA flow cytometry indicated that AMA slightly induced a G1 phase arrest of the cell cycle for 72 h. Treatment with 50 microM AMA induced apoptosis of approximately 17% in view of annexin V-staining cells. The dose of 50 microM AMA also induced loss of the mitochondrial membrane potential (DeltaPsi(m)) of approximately 38%. The intracellular reactive oxygen species (ROS) levels including O2(.-) were significantly increased in AMA-treated A549 cells. In conclusion, AMA inhibited the growth of A549 cells via inducing cell cycle arrest as well as triggering apoptosis. Growth inhibition in AMA-treated A549 cells was accompanied by an increase in ROS levels.

Arsenic trioxide (ATO) affects many biological processes such as cell proliferation, apoptosis, differentiation and angiogenesis. L-buthionine sulfoximine (BSO) is an inhibitor of GSH synthesis. We tested whether ATO reduced the viability of lung cancer A549 cells in vitro, and investigated the in vitro effect of the combination of ATO and BSO on cell viability in relation to apoptosis and the cell cycle. ATO caused a dose-dependant decrease of viability of A549 cells with an IC50 of more than 50 microM. Low doses of ATO or BSO (1 approximately 10 microM) alone did not induce cell death. However, combined treatment depleted GSH content and induced apoptosis, loss of mitochondrial transmembrane potential (DeltaPsi(m)) and cell cycle arrest in G2. Reactive oxygen species (ROS) increased or decreased depending on the concentration of ATO. In addition, BSO generally increased ROS in ATO-treated A549 cells. ROS levels were at least in part related to apoptosis in cells treated with ATO and/or BSO. In conclusion, we have demonstrated that A549 lung cells are very resistant to ATO, and that BSO synergizes with clinically achievable concentration of ATO. Our results suggest that combination treatment with ATO and BSO may be useful for treating lung cancer.

Antimycin A (AMA), an electron transport chain inhibitor in mitochondria can produce reactive oxygen species (ROS) in cells. It has been reported that ROS may have roles in cell cycle progression via regulating cell cycle-related proteins. In the present study, we investigated the changes of the cell cycle distribution in AMA-treated HeLa cells in relation to cell cycle-related proteins. DNA flow cytometric analysis indicated that treatment with AMA significantly induced an S phase arrest of the cell cycle at 72 h. AMA decreased the expression of cyclin-dependent kinase inhibitor (CDKI), p21 and p27, CDK4, and cdc2 proteins. The expression of CDK6, cyclin D1, cyclin E, cyclin A, and cyclin B proteins was increased by 0.5 microM AMA, but was decreased by 2 and 10 microM AMA. The phosphorylation of Rb on the Ser (780) residue was increased by 0.5 microM AMA. Furthermore, treatment with AMA caused the accumulation of cells expressing cyclin A, B, and D1 proteins at the S phase of the cell cycle. However, treatment with 100 microM AMA nonspecifically extended all phases of the cell cycle. In conclusion, treatment with AMA (2, 10 and 50 microM) induced an S phase arrest of the cell cycle. An S phase arrest was accompanied by the alteration of other cell cycle-regulated proteins as well as S phase-related proteins.

Arsenic trioxide (ATO) can affect many biological functions such as apoptosis and differentiation in various cells. We investigated the involvement of ROS and GSH in ATO-induced HeLa cell death using ROS scavengers, especially N-acetylcysteine (NAC). ATO increased intracellular O(2)(*-) levels and reduced intracellular GSH content. The ROS scavengers, Tempol, Tiron and Trimetazidine, did not significantly reduce levels of ROS or GSH depletion in ATO-treated HeLa cells. Nor did they reduce the apoptosis induced by ATO. In contrast, treatment with NAC reduced ROS levels and GSH depletion in the ATO-treated HeLa cells and prevented ATO-induced apoptosis. Treatment with exogenous SOD and catalase reduced the depletion of GSH content in ATO-treated cells. Catalase strongly protected the cells from ATO-induced apoptosis. In addition, treatment with SOD, catalase and NAC slightly inhibited the G1 phase accumulation induced by ATO. In conclusion, NAC protects HeLa cells from apoptosis induced by ATO by up-regulating intracellular GSH content and partially reducing the production of O(2)(*-).

Arsenic trioxide (ATO) affects many biological functions such as cell proliferation, apoptosis, differentiation and angiogenesis in various cells. We investigated the in vitro effects of ATO as a reactive oxygen species (ROS) generator or a glutathione (GSH) depletor on apoptosis in HeLa cells. ATO decreased the viability of HeLa cells in a dose-dependent manner with an IC50 of approximately 5-6 microM. ATO triggered apoptosis, which is accompanied by the loss of mitochondrial transmembrane potential (DeltaPsim). Intracellular general ROS levels in HeLa cells were increased or decreased depending on the concentration of ATO. Particularly, the levels of O2.- were increased by ATO. In addition, we detected a decreased GSH content in ATO-treated cells. The GSH-depleted cells mainly showed propidium iodine-positive staining, indicating that the majority of the cells were dead. Diethyldithiocarbamate (DDC; an inhibitor of Cu,Zn-SOD) induced apoptosis and the loss of mitochondrial transmembrane potential (DeltaPsim) in HeLa control cells. DDC intensified apoptosis, the loss of mitochondrial transmembrane potential, increased levels of O2.- and GSH depletion in ATO-treated cells. L-buthionine sulfoximine (BSO; an inhibitor of GSH synthesis) did not induce apoptosis in HeLa control cells, but increased levels of apoptosis, O2.- and GSH depletion in ATO-treated cells. In conclusion, the changes in intracellular GSH levels rather than ROS levels are tightly related to the enhancement of ATO-induced apoptosis in HeLa cells by DDC or BSO.

Pyrogallol, a polyphenol, is known to be a superoxide anion (O2(.-)) generator. We investigated the involvement of glutathione (GSH) and reactive oxygen species (ROS) in pyrogallol-induced HeLa cell death. We measured the changes of ROS levels, GSH levels, sub-G1 cells, annexin V/PI staining cells and mitochondria membrane potential (DeltaPsi m) in HeLa cells treated with pyrogallol and/or ROS scavenger. The intracellular ROS levels were decreased or increased depending on the concentration of pyrogallol. The level of O2(.-) was significantly increased and superoxide dismutase (SOD) activity was down-regulated by pyrogallol. Pyrogallol reduced intracellular GSH content in HeLa cells. The ROS scavengers, Tempol, Tiron, Trimetazidine and N-acetylcysteine (NAC), did not down-regulate the production of O2(.-). However, treatment with NAC showed the recovery of GSH depletion and significantly rescued cells from pyrogallol-induced apoptosis. In addition, the recovery of GSH depletion by SOD and catalase was accompanied by the decrease of apoptosis levels. Furthermore, NAC and SOD significantly inhibited CMF-negative (GSH-depleted) and PI-positive cells induced by pyrogallol. Taken together, pyrogallol potently increased intracellular O2(.-) levels and decreased GSH content in HeLa cells, and NAC, SOD and catalase significantly rescued HeLa cells from pyrogallol-induced apoptosis accompanied by the recovery of GSH depletion.

ADP-ribosyl cyclase (ADPR-cyclase) produces a Ca(2+)-mobilizing second messenger cyclic ADP-ribose (cADPR) from NAD(+). In this study, we investigated the molecular basis of ADPR-cyclase activation and the following cellular events in angiotensin II (ANG II) signaling in mouse mesangial cells (MMCs). Treatment of MMCs with ANG II induced an increase in intracellular Ca(2+) concentrations through a transient Ca(2+) release via an inositol 1,4,5-trisphosphate receptor and a sustained Ca(2+) influx via L-type Ca(2+) channels. The sustained Ca(2+) signal, but not the transient Ca(2+) signal, was blocked by a cADPR antagonistic analog, 8-bromo-cADPR (8-Br-cADPR), and an ADPR-cyclase inhibitor, 4,4'-dihydroxyazobenzene (DHAB). In support of the results, ANG II stimulated cADPR production in a time-dependent manner, and DHAB inhibited ANG II-induced cADPR production. Application of pharmacological inhibitors revealed that activation of ADPR-cyclase by ANG II involved ANG II type 1 receptor, phosphoinositide 3-kinase, protein tyrosine kinase, and phospolipase C-gamma1. Moreover, DHAB as well as 8-Br-cADPR abrogated ANG II-mediated Akt phosphorylation, nuclear translocation of nuclear factor of activated T cell, and uptake of [(3)H]thymidine and [(3)H]leucine in MMCs. These results demonstrate that ADPR-cyclase in MMCs plays a pivotal role in ANG II signaling for cell proliferation and protein synthesis.

Antimycin A (AMA) inhibits succinate oxidase and NADH oxidase, and also inhibits mitochondrial electron transport between cytochromes b and c. We investigated the involvement of ROS and GSH in AMA-induced HeLa cell death. AMA increased the intracellular H(2)O(2) and O(2)(*-) levels and reduced the intracellular GSH content. ROS scavengers (Tempol, Tiron, Trimetazidine and NAC) did not down-regulate the production of ROS and inhibit apoptosis in AMA-treated cells. Treatment with NAC and N-propylgallate showing the enhancement of GSH depletion in AMA-treated cells significantly intensified the levels of apoptosis. Calpain inhibitors I and II (calpain inhibitor III) and Ca(2+)-chelating agent (EGTA/AM) significantly reduced H(2)O(2) levels in AMA-treated HeLa cells. However, treatment with calpain inhibitor III intensified the levels of O(2)(*-) in AMA-treated cells. In addition, calpain inhibitor III strongly depleted GSH content with an enhancement of apoptosis in AMA-treated cells. Conclusively, the changes of ROS by AMA were not tightly correlated with apoptosis in HeLa cells. However, intracellular GSH levels are tightly related to AMA-induced cell death.

We investigated whether alpha-lipoic acid (alpha-LA), an antioxidant, attenuates the ischemia-reperfusion (I/R)-induced dysregulation of these transporters. Both renal pedicles of male Sprague-Dawley rats were clamped for 40 min. alpha-LA (80 mg/kg) was administered intraperitoneally before and immediately after induction of ischemia. After 2 days, the expression of aquaporins (AQPs), sodium transporters, and nitric oxide synthases (NOS) was determined in the kidney by immunoblotting and immunohistochemistry. The expression of endothelin-1 (ET-1) mRNA was determined by real-time PCR. Activities of adenylyl cyclase and guanylyl cyclase were measured by stimulated generation of cAMP and cGMP, respectively. The expression of AQP1-3 as well as that of the alpha(1)-subunit of Na-K-ATPase, type 3 Na/H exchanger, Na-K-2Cl cotransporter, and Na-Cl cotransporter was markedly decreased in response to I/R. The expression of type VI adenylyl cyclase was decreased in I/R-injured rats, which was counteracted by the treatment of alpha-LA. AVP-stimulated cAMP generation was blunted in I/R rats and was then ameliorated by alpha-LA treatment. alpha-LA treatment attenuated the downregulation of AQPs and sodium transporters. The expression of endothelial NOS was decreased in I/R rats, which was prevented by alpha-LA. The cGMP generation in response to sodium nitroprusside was blunted in I/R rats, which was also significantly prevented by alpha-LA. The mRNA expression of ET-1 was increased, which was recovered to the control level by alpha-LA treatment. In conclusion, alpha-LA treatment prevents I/R-induced dysregulation of AQPs and sodium transporters in the kidney, possibly through preserving normal activities of local AVP/cAMP, nitric oxide/cGMP, and ET systems.

We investigated the in vitro effects of arsenic trioxide on cell growth, cell cycle regulation, and apoptosis in As4.1 juxtaglomerular cells. Arsenic trioxide inhibited the growth of As4.1 cells with an IC(50) of approximately 5 microM. Arsenic trioxide induced S phase arrest of the cell cycle and very efficiently stimulated apoptosis in As4.1 cells, as evidenced by flow cytometric detection of sub-G(1) DNA content, annexin V binding assay, and 4'-6-diamidino-2-phenylindole staining. This apoptotic process was accompanied by the loss of mitochondrial transmembrane potential (DeltaPsi(m)), a decrease in Bcl-2, the activation of caspase-3, and cleavage of poly(ADP-ribose) polymerase. However, all of the caspase inhibitors tested in this experiment failed to rescue As4.1 cells from arsenic trioxide-induced cell death in view of sub-G(1) cells and annexin V positive-staining cells. However, a caspase-8 inhibitor (Z-IETD-FMK) noticeably decreased the loss of DeltaPsi(m) in arsenic trioxide-treated cells. When we examined the changes in reactive oxygen species (ROS), H(2)O(2), or O(2)(*-) in arsenic trioxide-treated cells, H(2)O(2) was significantly decreased and O(2)(*-) was increased. In addition, we detected a decreased GSH content in arsenic trioxide-treated cells. Taken together, we have demonstrated that arsenic trioxide as a ROS generator potently inhibited the growth of As4.1 JG cells through S phase arrest of the cell cycle and caspase-independent apoptosis.

Adenosine acts as an important protector of ischemic myocardium through coronary vasodilation and the depression of cardiac contractility. The protective effect of adenosine may partly relate to the cardiac hormone atrial natriuretic peptide (ANP). The aim of the present study was to investigate the effects of adenosine and the adenosine receptor subtype on atrial hemodynamics and ANP release using isolated perfused beating rat atria. Adenosine, a nonselective adenosine receptor agonist, increased the ANP release with negative inotropism in a dose-dependent manner. Adenosine-stimulated ANP release was attenuated by a selective A1 antagonist but not A(2A) antagonist or A3 antagonist. The order of potency of the various agonists for the ANP release was A1 agonists>>A3 agonist=adenosine>A(2A) agonist. The order of potency for the negative inotropy was A1 agonists>adenosine=A(2A) agonist>A3 agonist. The negative inotropism and ANP release by a specific A1 agonist (N6-cyclopentyl-adenosine) were also attenuated by A1 antagonist but not A(2A) antagonist or A3 antagonist. Treatment with A1 agonist resulted in a decrease of cAMP contents in atria and perfusates. The agonist-stimulated ANP release was significantly attenuated in the presence of forskolin, isoproterenol 8-Br-cAMP, or an adenylyl cyclase inhibitor. These results suggest that the A1 receptor subtype is responsible for the adenosine-induced ANP release and negative inotropism through adenylyl cyclase-cAMP pathway.

Extracellular ATP acts as a local regulator of physiological functions in the cardiovascular system via P1 and P2 receptors. However, little is known about the effect of ATP on the release of atrial natriuretic peptide (ANP) secretion. The purpose of this study was to investigate the effects of extracellular ATP on atrial hemodynamics and ANP release and to identify their receptor-mediated mechanism. ATP was infused into isolated perfused beating rat atria in the absence and presence of various receptor antagonists. ATP (from 0.1 to 30 microM) increased the ANP release with negative inotropism in a dose-dependent manner. ADP (30 microM) also caused an increase in ANP release with similarity to ATP, but alpha,beta-methylene ATP (alpha,beta-MeATP, P2X1 receptor agonist) and 2-methylthioADP (2-MesADP, P2Y1 receptor agonist) did not. The rank order of potency for the increment of ANP release was adenosine>ATP=ADP>2-MesADP>alpha,beta-MeATP. In contrast, UTP, an agonist for P2Y2,4,6 receptor, caused a decrease in ANP release without changes in contractility. Extracellular ATP-induced increase in ANP release and negative inotropism were completely blocked by the pretreatment of 8-cyclopentyl-1,3-dipropylxanthine (P1 receptor antagonist), but not by pyridoxal phosphate-6-azobenzene-2,4-disulfonic acid (P2X1 receptor antagonist) and suramin (P2XY receptor antagonist). Reactive Blue 2 (P2Y receptor antagonist) caused an augmentation of ATP-induced increase in ANP release without affecting negative inotropism. Adenosine 5'-(alpha,beta-methylene) diphosphate, an ectonucleotidase inhibitor, did not affect ATP-induced augmentation of ANP release with negative inotropy. These results suggest that extracellular ATP-induced increase in ANP release and negative inotropism are mediated mainly by P1 receptor, and UTP decreases ANP release. Therefore, we suggest that extracellular ATP and UTP may have opposite actions on the regulation of ANP secretion.

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.

Changes in cyclic nucleotide production and atrial dynamics have been known to modulate atrial natriuretic peptide (ANP) release. Although cardiac atrium expresses histamine receptors and contains histamine, the role of histamine in the regulation of ANP release has to be defined. The purpose of the present study was to define the effect of histamine on the regulation of ANP release in perfused beating rabbit atria. Histamine decreased ANP release concomitantly with increases in cAMP efflux and atrial dynamics in a concentration-dependent manner. Histamine-induced decrease in ANP release was a function of an increase in cAMP production. Blockade of histamine H2 receptor with cimetidine but not of H1 receptor with triprolidine abolished the responses of histamine. Cell-permeable cAMP analog, 8-Br-cAMP, mimicked the effects of histamine, and the responses were dose-dependent and blocked by a protein kinase A (PKA)-selective inhibitor, KT5720. Nifedipine failed to modulate histamine-induced decrease in ANP release. Protein kinase nonselective inhibitor staurosporine blocked histamine-induced changes in a concentration-dependent manner. KT5720 and RP-adenosine 3',5'-cyclic monophosphorothioate, another PKA-selective inhibitor, attenuated histamine-induced changes. These results suggest that histamine decreases atrial ANP release by H2 receptor-cAMP signaling via PKA-dependent and -independent pathways.

The myocardial interstitium is important in regulating cardiac function. Between the atrial lumen and the pericardial space are transmural pathways, and movement of interstitial fluid (ISF) through these pathways is one of the main driving forces regulating translocation of substances from the interstitium into the blood. To define how ISF translocation from the interstitial space into the luminal space is regulated by each component of atrial hemodynamics, we devised a new rabbit atrial model in which each physical parameter could be controlled independently. Using this system, we also defined the physiological role of the cardiac Na(+)/Ca(2+) exchanger on secretion of atrial natriuretic peptide (ANP) by depletion of extracellular Na(+) ([Na(+)](o)). Increases in stroke volume and atrial end-systolic volume increased ISF translocation and ANP secretion. However, an increase in atrial rate did not influence ISF translocation but, rather, increased ANP secretion. Gradual depletion of [Na(+)](o) caused gradual increases in ANP secretion and intracellular Ca(2+) ([Ca(2+)](i)), which were blocked in the presence of Ca(2+)-free buffer and Ni(2+), but not in the presence of KB-R7943, diltiazem, mibefradil, caffeine, or monensin. Amiloride and its analog blocked an increase in ANP secretion but not an increase in [Ca(2+)](i) by [Na(+)](o) depletion. Therefore, we suggest that ANP secretion and ISF translocation may be differently controlled by each physical factor. These results also suggest that the increase in ANP secretion in response to [Na(+)](o) depletion may involve inhibition of Na(+)/Ca(2+) and Na(+)/H(+) exchangers but not an increase in [Ca(2+)](i).

To define developmental changes in atrial natriuretic peptide (ANP) secretion and in the cross talk between C-type natriuretic peptide (CNP) and ANP, we performed experiments in isolated perfused nonbeating cardiac atria isolated from rabbits between 1 and 8 wk of age. Changes in atrial pressure resulted in increases in atrial volume that rose with age and reached the peak value at 4 wk. A rise in volume change increased ANP secretion with concomitant translocation of extracellular fluid (ECF) into the atrial lumen, which increased with age and reached the peak value at 4 wk. The positive relationship between stretch-induced ANP secretion and ECF translocation shifted upward and leftward with age. CNP suppressed stretch-induced ANP secretion in the 8-wk-old group but not in the 2- and 4-wk-old groups without differences in ECF translocation and atrial volume. Therefore, the ANP secretion in terms of ECF translocation was markedly suppressed by CNP in the 8-wk-old group but not in the 2- and 4-wk-old groups. The production of cGMP by CNP in atrial tissue membranes was markedly attenuated in young rabbits. However, 8-bromo-cGMP suppressed stretch-induced ANP secretion in 2- and 8-wk-old groups. Natriuretic peptide receptor-B mRNA was similar in both groups. Therefore, we conclude that the inhibitory effect of CNP on atrial ANP secretion is developmentally regulated, being absent during normal cardiac development in young animals and intact in adult animals.