atrial-natriuretic-factor and chelerythrine

It has been shown that, in the remote myocardium after infarction (MI), protein kinase C (PKC) inhibition reduces apoptosis both by blocking proapoptotic pathways and by activating antiapoptotic signals including the Akt pathway. However, it was open if vice versa, blockade of antiapoptotic pathways may influence proapoptotic signals. To clarify this, the present study tested the effects of the PI3-kinase blocker Wortmannin on proapoptotic signals and on apoptosis execution in the remote myocardium after infarction. Rats were subjected to MI by LAD ligation in situ. Some were pre-treated with Wortmannin alone or in combination with the PKC inhibitor Chelerythrine. After 24 h, pro- and anti-apoptotic signals (caspase-3, PKC isoforms, p38-MAPK, p42/44-MAPK, Akt, Bad), and marker of apoptosis execution (TUNEL) were quantified in the myocardium remote from the infarction. Wortmannin treatment increased apoptosis in the remote myocardium both at baseline and after MI, together with an activation of the PKC-δ/p38-MAPK-pathway. PKC-ε and p42/44-MAPK were unaffected. Combined treatment with Wortmannin and Chelerythrine fully reversed the pro-apoptotic effects of Wortmannin both at baseline and after MI. The PKC-δ-p38-MAPK-pathway as a strong signal for apoptosis in the non-infarcted myocardium can be influenced by targeting the anti-apoptotic PI3-kinase pathway. This gives evidence of a bi-directional crosstalk of pro- and anti-apoptotic signals after infarction.

Topics: Androstadienes; Animals; Apoptosis; Atrial Natriuretic Factor; Benzophenanthridines; Biphenyl Compounds; Caspase 3; Coronary Vessels; Enzyme Induction; Irbesartan; Isoenzymes; Ligation; Male; MAP Kinase Signaling System; Myocardial Infarction; Myocardium; p38 Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Kinase C-delta; Protein Kinase C-epsilon; Protein Precursors; Protein Processing, Post-Translational; Rats; Rats, Wistar; Tetrazoles; Wortmannin

Apoptotic processes may be implicated in the molecular pathomechanisms of ventricular remodeling after myocardial infarction (MI). The modulation of apoptosis by pro- and anti-apoptotic pathways in the myocardium remote from the infarction, including its link to protein kinase C (PKC), was focus of the present study.. Rats were subjected to MI by LAD ligation in situ. Some animals were pretreated with the PKC inhibitor chelerythrine. After 1 h up to 28 days, pro- and anti-apoptotic signals (caspase-3, Bcl-2/Bax ratio, Akt, Bad), and marker of apoptosis execution (DNA laddering, TUNEL) were quantified in the myocardium remote from the infarction.. Activation of caspase-3, a pro-apoptotic shift of the Bcl-2/Bax ratio, and DNA fragmentation were observed as early as 3 h after infarction and persisted up to 28 days. Akt- and Bad-phosphorylation was unchanged. Chelerythrine markedly reduced DNA fragmentation. Caspase-3 activation was unchanged. Surprisingly, Bad and Akt phosphorylation were highly increased (180% and 750% of control).. Chelerythrine influences the balance of pro- and anti-apoptotic pathways in the remote myocardium after infarction, with an inhibition of proapoptotic and an activation of anti-apoptotic signals.

Topics: Animals; Apoptosis; Apoptosis Inducing Factor; Atrial Natriuretic Factor; bcl-Associated Death Protein; Benzophenanthridines; Biomarkers; Disease Models, Animal; DNA Fragmentation; In Situ Nick-End Labeling; In Vitro Techniques; Male; Models, Biological; Myocardial Contraction; Myocardial Infarction; Myocardium; Phosphorylation; Protein Kinase C-delta; Proto-Oncogene Proteins c-akt; Rats; Rats, Wistar; Signal Transduction; Time Factors

Yeast silent information regulator 2 (Sir2), a nicotinamide adenine dinucleotide-dependent histone deacetylase (HDAC) and founding member of the HDAC class III family, functions in a wide array of cellular processes, including gene silencing, longevity, and DNA damage repair. We examined whether or not the mammalian ortholog Sir2 affects growth and death of cardiac myocytes. Cardiac myocytes express Sir2alpha predominantly in the nucleus. Neonatal rat cardiac myocytes were treated with 20 mmol/L nicotinamide (NAM), a Sir2 inhibitor, or 50 nmol/L Trichostatin A (TSA), a class I and II HDAC inhibitor. NAM induced a significant increase in nuclear fragmentation (2.2-fold) and cleaved caspase-3, as did sirtinol, a specific Sir2 inhibitor, and expression of dominant-negative Sir2alpha. TSA also modestly increased cell death (1.5-fold) but without accompanying caspase-3 activation. Although TSA induced a 1.5-fold increase in cardiac myocyte size and protein content, NAM reduced both. In addition, NAM caused acetylation and increases in the transcriptional activity of p53, whereas TSA did not. NAM-induced cardiac myocyte apoptosis was inhibited in the presence of dominant-negative p53, suggesting that Sir2alpha inhibition causes apoptosis through p53. Overexpression of Sir2alpha protected cardiac myocytes from apoptosis in response to serum starvation and significantly increased the size of cardiac myocytes. Furthermore, Sir2 expression was increased significantly in hearts from dogs with heart failure induced by rapid pacing superimposed on stable, severe hypertrophy. These results suggest that endogenous Sir2alpha plays an essential role in mediating cell survival, whereas Sir2alpha overexpression protects myocytes from apoptosis and causes modest hypertrophy. In contrast, inhibition of endogenous class I and II HDACs primarily causes cardiac myocyte hypertrophy and also induces modest cell death. An increase in Sir2 expression during heart failure suggests that Sir2 may play a cardioprotective role in pathologic hearts in vivo.

Topics: Acetylation; Alkaloids; Animals; Apoptosis; Atrial Natriuretic Factor; Benzamides; Benzophenanthridines; Cell Nucleus; Cell Size; Cell Survival; Cells, Cultured; Culture Media, Serum-Free; Cysteine Proteinase Inhibitors; Dogs; Gene Silencing; Genes, Dominant; Genes, p53; Heart Failure; Heart Ventricles; Hydroxamic Acids; Hypertrophy; Hypertrophy, Left Ventricular; Longevity; Mice; Myocytes, Cardiac; Naphthols; Niacinamide; Phenanthridines; Protein Processing, Post-Translational; Rats; Rats, Wistar; Recombinant Fusion Proteins; Sirtuin 1; Sirtuins; Transcription, Genetic; Tumor Suppressor Protein p53

Carperitide, a synthetic alpha-human atrial natriuretic peptide (ANP) is a newly developed drug for the treatment of heart failure. However, effects of carperitide on susceptibility to ischemia reperfusion injury are left to be determined. Isolated rat hearts were subjected to Langendorff perfusion. Six hearts received 0.1 microM of carperitide for 10 min, 6 hearts received 1 mM of a NO synthetase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) for 5 min before the infusion of carperitide, 6 hearts received 0.02 microM of a PKC synthetase inhibitor chelerythrine chloride for 5 min before the infusion of carperitide, 6 hearts received 100 microM of a selective mitochondrial ATP-sensitive potassium (KATP) channel blocker 5-dehydroxydecanoate (5HD) before the infusion of carperitide, 6 hearts received 10 microM of a soluble guanylate cyclase inhibitor methylene blue for 5 min before the infusion of carperitide, and 6 hearts served as a control with no drug infusion. All hearts were then subjected to 20 min of global ischemia followed by 120 min of reperfusion. Left ventricular pressures and coronary flow were measured throughout the experiment and infarct size was detected at the end of experiment. Both plasma and tissue cGMP levels were also determined. The results showed: (1) Carperitide significantly reduced infarct size compared to control (26.1 +/- 2.8 vs. 42.7 +/- 2.3%, carperitide vs. control, p < 0.05). This effect was reversed by L-NAME, chelerythrine and 5HD, but not methylene blue. (2) Plasma cGMP levels were increased in carperitide-treated group. This effect was reversed by L-NAME (0.16 +/- 0.03 vs. 1.04 +/- 0.09* vs. 0.28 +/- 0.02 nmol/L, control vs. carperitide vs. L-NAME, *p < 0.01 vs. control). We conclude that preischemic infusion of carperitide exerts cardioprotective effects possibly through NO-PKC dependent pathway followed by mitochondrial KATP channel activation.

Topics: Adenosine Triphosphate; Alkaloids; Animals; Atrial Natriuretic Factor; Benzophenanthridines; Cyclic GMP; Enzyme Inhibitors; Heart; Ischemia; Ischemic Preconditioning, Myocardial; Methylene Blue; Myocardial Ischemia; Myocardial Reperfusion; Myocardial Reperfusion Injury; Myocardium; NG-Nitroarginine Methyl Ester; Nitric Oxide; Phenanthridines; Potassium Channel Blockers; Potassium Channels; Protein Kinase C; Rats; Reperfusion; Time Factors; Tissue Distribution

Serotonin (5-hydroxytryptamine [5-HT]) receptors are located in peripheral tissues as well as in the central nervous system. Serotonin receptors mediate positive inotropic and chronotropic effects in atria. The aim of this study was to investigate physiological role of endogenous serotonin on the regulation of atrial natriuretic peptide (ANP) secretion from the atria.. An isolated perfused nonbeating rat atrial model was used. Changes in atrial volume induced by increasing intra-atrial pressure were measured. The concentration of ANP was measured by radioimmunoassay and the translocation of ECF was measured by [3H]-inulin clearance.. Serotonin, an endogenous 5-HT receptor agonist, caused concentration-dependent suppressions of stretch-induced ANP secretion, which were less pronounced than those caused by alpha-methyl-5-HT maleate, a 5-HT(2) receptor selective agonist. The suppression of stretch-induced ANP secretion due to serotonin and alpha-methyl-5-HT maleate was attenuated by ketanserin, a 5-HT(2) receptor antagonist, and accentuated by RS23597-190, a 5-HT(4) receptor antagonist. The suppressive effect of serotonin on ANP secretion was attenuated by neomycin, staurosporine, and chelerythrine. In contrast, 2-[1-(4-piperonyl)piperazinyl]benzothiazole, a 5-HT(4) receptor selective agonist, caused an accentuation of stretch-induced ANP secretion, which was completely blocked by RS23597-190 and SB203186 HCl but not by ketanserin. This effect was not affected by MDL12330, KT-5720, or H-89. The intracellular Ca(2+) concentration in single atrial myocytes was not changed by serotonin and agonist for either 5-HT(2) or 5-HT(4) receptor.. These results suggest that atrial 5-HT(2) and 5-HT(4) receptor agonists have opposite actions on the regulation of ANP secretion and the suppressive effect of serotonin on the ANP secretion may act through 5-HT(2) receptor and phospholipase C pathway.

Topics: Adenylyl Cyclase Inhibitors; Alkaloids; Aminobenzoates; Animals; Atrial Natriuretic Factor; Benzophenanthridines; Benzothiazoles; Calcium; Carbazoles; Cyclic AMP-Dependent Protein Kinases; Depression, Chemical; Dose-Response Relationship, Drug; Heart; Heart Atria; Imines; Indoles; Isoquinolines; Ketanserin; Male; Myocytes, Cardiac; Neomycin; para-Aminobenzoates; Perfusion; Phenanthridines; Piperazines; Piperidines; Protein Kinase C; Pyrroles; Rats; Rats, Sprague-Dawley; Receptors, Serotonin; Receptors, Serotonin, 5-HT4; Serotonin; Serotonin Antagonists; Serotonin Receptor Agonists; Staurosporine; Sulfonamides; Thiazoles; Type C Phospholipases

The mechanism of endothelin-1 (ET-1)-induced atrial natriuretic peptide (ANP) release was studied in neonatal rat ventricular cardiomyocytes. These cells expressed a single high-affinity class of ETA receptor (dissociation constant = 54 +/- 18 pM, n = 3), but no ETB receptors. Incubation of cardiomyocytes with ET-1 led to concentration-dependent ANP release and prostacyclin production. ET-1-induced ANP release was affected by neither protein kinase C (PKC) inhibition or downregulation nor by cyclooxygenase inhibition, indicating that ET-1-stimulated ANP secretion is not a PKC-mediated, prostaglandin-dependent process. Furthermore, ET-1 significantly stimulated adenosine 3',5'-cyclic monophosphate (cAMP) production and increased cytosolic calcium concentration in these preparations. Both ET-1-induced calcium influx and ANP release were decreased by the cAMP antagonist Rp-cAMPS, the Rp diastereoisomer of cAMP. Moreover, ET-1-induced ANP secretion was strongly inhibited in the presence of nifedipine as well as in the absence of extracellular calcium. Thus our results suggest that ET-1 stimulates ANP release in ventricular cardiomyocytes via an ETA receptor-mediated pathway involving cAMP formation and activation of a nifedipine-sensitive calcium channel.

Topics: Alkaloids; Animals; Animals, Newborn; Atrial Natriuretic Factor; Benzophenanthridines; Calcium; Cells, Cultured; Cyclic AMP; Cyclooxygenase Inhibitors; Egtazic Acid; Endothelin-1; Enzyme Inhibitors; Epoprostenol; Heart; Heart Ventricles; Kinetics; Myocardium; Naphthalenes; Nifedipine; Phenanthridines; Protein Kinase C; Rats; Rats, Wistar; Receptor, Endothelin A; Receptor, Endothelin B; Receptors, Endothelin; Staurosporine; Thionucleotides

To characterize the effects of the cellular events associated with contraction on atrial natriuretic factor (ANF) secretion, primary neonatal rat atrial myocytes were electrically paced to contract while being monitored for ANF release, cytoplasmic calcium, phosphoinositide hydrolysis, and protein kinase C activation. Similar measurements were also carried out in the presence of endothelin-1 (ET) for comparison of contraction-related and hormone-stimulated ANF secretion. Pacing (6-8 Hz) immediately increased ANF secretion by 3-5-fold and the time-averaged cytoplasmic calcium concentration (as monitored with indo-1 fluorescence) varied with pace frequency in a similar manner, suggesting that cytoplasmic calcium may play a key role in pace-induced ANF secretion. Furthermore, nifedipine and ryanodine, which inhibited the contractile calcium transients, inhibited pace-induced ANF release, whereas Bay K 8644 increased both the calcium transients and ANF secretion. Pace-induced ANF release was also completely inhibited by KN-62, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMK) but was not inhibited by chelerythrine, a protein kinase C-selective inhibitor. Pace-induced ANF release averaged 40% of that elicited by ET which is known to require both PKC and CaMK for maximal effects on ANF secretion. The effects of pacing and ET on ANF secretion were approximately additive. In contrast to pacing, ET strongly stimulated phosphoinositide hydrolysis, activated PKC, and did not increase cytoplasmic calcium. Thus, regulation of ANF secretion by contraction rate depends primarily on the contractile calcium transients and CaMK and is independent of PKC.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Alkaloids; Analysis of Variance; Animals; Animals, Newborn; Atrial Natriuretic Factor; Benzophenanthridines; Calcium; Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; Cytoplasm; Endothelins; Fluorescent Dyes; Heart; Heart Atria; Indoles; Isoquinolines; Kinetics; Myocardial Contraction; Myocardium; Phenanthridines; Phorbol 12,13-Dibutyrate; Phosphatidylinositols; Phosphoproteins; Piperazines; Protein Kinase C; Rats; Rats, Sprague-Dawley; Spectrometry, Fluorescence

The protease, alpha-thrombin (alpha Th), affects myocardial cell contractility, a feature common among agents that induce hypertrophy. However, it is not known whether cardiac myocytes possess alpha Th receptors (alpha Th-R), or if long term treatment with alpha Th can enhance growth and gene expression. In the present study primary neonatal rat ventricular myocytes expressed a 3.6-kilobase mRNA species that hybridized with a rat alpha Th-R-specific probe. After 48 h, alpha Th induced hypertrophy, sarcomeric organization, and enhanced atrial natriuretic factor (ANF) expression, all of which were blocked by the alpha Th-selective protease inhibitor, D-Phe-Pro-Arg-chloromethyl ketone. The alpha Th-R agonist peptide, SFLLRNPND, was a potent activator of ANF expression, however, the non-agonist, FLLRNPND, was inactive. Transfection experiments showed the enhancement of ANF expression by alpha Th to be transcriptional. The abilities of alpha Th to induce myocyte hypertrophy and to augment ANF transcription and peptide production were inhibited by the protein kinase C inhibitor, chelerythrine, and by the tyrosine kinase inhibitor, tyrphostin. Thus, myocardial cell alpha Th-Rs are stimulated by the specific proteolytic actions of alpha Th, and pathways involving both protein kinase C and protein tyrosine kinases are required for subsequent hypertrophy and ANF expression. Further, these findings suggest a new role for extracellular proteases as regulators of myocardial cell gene expression and growth.

Topics: Alkaloids; Amino Acid Sequence; Animals; Animals, Newborn; Atrial Natriuretic Factor; Benzophenanthridines; Blotting, Northern; Cardiomegaly; Catechols; Cells, Cultured; Gene Expression; Heart Ventricles; Kinetics; Molecular Sequence Data; Myocardium; Nitriles; Oligopeptides; Phenanthridines; Platelet Aggregation Inhibitors; Polymerase Chain Reaction; Protease Inhibitors; Protein-Tyrosine Kinases; Radioimmunoassay; Rats; Receptors, Cell Surface; Receptors, Thrombin; RNA, Messenger; Thrombin; Tyrphostins