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1.
J Biol Chem ; 278(28): 25542-7, 2003 Jul 11.
Article in English | MEDLINE | ID: mdl-12721309

ABSTRACT

Oxidative stress promotes cardiac myocyte apoptosis through the mitochondrial death pathway. Since Bcl-2 family proteins are key regulators of apoptosis, we examined the effects of H2O2 on the expression of principal Bcl-2 family proteins (Bcl-2, Bcl-xL, Bax, Bad) in neonatal rat cardiac myocytes. Protein expression was assessed by immunoblotting. Bcl-2, Bax, and Bad were all down-regulated in myocytes exposed to 0.2 mm H2O2, a concentration that induces apoptosis. In contrast, although Bcl-xL levels initially declined, the protein was re-expressed from 4-6 h. Bcl-xL mRNA was up-regulated from 2 to 4 h in neonatal rat or mouse cardiac myocytes exposed to H2O2, consistent with the re-expression of protein. Four different untranslated first exons have been identified for the Bcl-x gene (exons 1, 1B, 1C, and 1D, where exon 1 is the most proximal and exon 1D the most distal to the coding region). All were detected in mouse or rat neonatal cardiac myocytes, but exon 1D was not expressed in adult mouse hearts. In neonatal mouse or rat cardiac myocytes, H2O2 induced the expression of exons 1B, 1C, and 1D, but not exon 1. These data demonstrate that the Bcl-x gene is selectively responsive to oxidative stress, and the response is mediated through distal promoter regions.


Subject(s)
Hydrogen Peroxide/pharmacology , Myocardium/metabolism , Proto-Oncogene Proteins c-bcl-2/biosynthesis , Proto-Oncogene Proteins c-bcl-2/genetics , Animals , Apoptosis , Cells, Cultured , DNA Primers/pharmacology , DNA, Complementary/metabolism , Down-Regulation , Exons , Immunoblotting , Mice , Mice, Inbred C57BL , Models, Genetic , Oxidative Stress , Promoter Regions, Genetic , RNA/metabolism , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Spleen/metabolism , Time Factors , Up-Regulation , bcl-X Protein
2.
Pharmacol Ther ; 97(3): 223-61, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12576135

ABSTRACT

Cardiac myocyte death, whether through necrotic or apoptotic mechanisms, is a contributing factor to many cardiac pathologies. Although necrosis and apoptosis are the widely accepted forms of cell death, they may utilize the same cell death machinery. The environment within the cell probably dictates the final outcome, producing a spectrum of response between the two extremes. This review examines the probable mechanisms involved in myocyte death. Caspases, the generally accepted executioners of apoptosis, are significant in executing cardiac myocyte death, but other proteases (e.g., calpains, cathepsins) also promote cell death, and these are discussed. The two principal cell death pathways (death receptor- and mitochondrial-mediated) are described in relation to the emerging structural information for the principal proteins, and they are discussed relative to current understanding of myocyte cell death mechanisms. Whereas the mitochondrial pathway is probably a significant factor in myocyte death in both acute and chronic phases of myocardial diseases, the death receptor pathway may prove significant in the longer term. The Bcl-2 family of proteins are key regulators of the mitochondrial death pathway. These proteins are described and their possible functions are discussed. The commitment to cell death is also influenced by protein kinase cascades that are activated in the cell. Whereas certain pathways are cytoprotective (e.g., phosphatidylinositol 3'-kinase), the roles of other kinases are less clear. Since myocyte death is implicated in a number of cardiac pathologies, attenuation of the death pathways may prove important in ameliorating such disease states, and possible therapeutic strategies are explored.


Subject(s)
Myocytes, Cardiac/cytology , Myocytes, Cardiac/physiology , Amino Acid Sequence/physiology , Animals , Caspases/genetics , Caspases/metabolism , Cell Death/drug effects , Cell Death/physiology , Cell Survival/drug effects , Cell Survival/physiology , Enzyme Activation/drug effects , Enzyme Activation/physiology , Humans , Molecular Sequence Data , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/enzymology , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Sequence Homology, Amino Acid
3.
J Mol Cell Cardiol ; 34(7): 749-63, 2002 Jul.
Article in English | MEDLINE | ID: mdl-12099715

ABSTRACT

Studies in non-cardiomyocytic cells have shown that phosphorylation of the Bcl-2 family protein Bad on Ser-112, Ser-136 and Ser-155 decreases its pro-apoptotic activity. Both phenylephrine (100 microM) and the cell membrane-permeating cAMP analog, 8-(4-chlorophenylthio)-cAMP (100 microM), protected against 2-deoxy-D-glucose-induced apoptosis in neonatal rat cardiac myocytes as assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). In cardiac myocytes, phenylephrine primarily stimulates the alpha-adrenoceptor, but, at high concentrations (100 microM), it also increases the activity of the cAMP-dependent protein kinase, protein kinase A (PKA) through the beta-adrenoceptor. Phenylephrine (100 microM) promoted rapid phosphorylation of Bad(Ser-112) and Bad(Ser-155), though we were unable to detect phosphorylation of Bad(Ser-136). Phosphorylation of Bad(Ser-112) was antagonized by either prazosin or propranolol, indicating that this phosphorylation required stimulation of both alpha(1)- and beta-adrenoceptors. Phosphorylation of Bad(Ser-155) was antagonized only by propranolol and was thus mediated through the beta-adrenoceptor. Inhibitor studies and partial purification of candidate kinases by fast protein liquid chromatography showed that the p90 ribosomal S6 kinases, p90RSK2/3 [which are activated by the extracellular signal-regulated kinases 1 and 2 (ERK1/2)] directly phosphorylated Bad(Ser-112), whereas the PKA catalytic subunit directly phosphorylated Bad(Ser-155). However, efficient phosphorylation of Bad(Ser-112) also required PKA activity. These data suggest that, although p90RSK2/3 phosphorylate Bad(Ser-112) directly, phosphorylation of this site is enhanced by phosphorylation of Bad(Ser-155). These phosphorylations potentially diminish the pro-apoptotic activity of Bad and contribute to the cytoprotective effects of phenylephrine and 8-(4-chlorophenylthio)-cAMP.


Subject(s)
Carrier Proteins/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Mitogen-Activated Protein Kinases/metabolism , Myocytes, Cardiac/metabolism , Phenylephrine/pharmacology , Animals , Apoptosis/physiology , Myocytes, Cardiac/drug effects , Phosphorylation/drug effects , Rats , Rats, Sprague-Dawley , Receptors, Adrenergic/metabolism , bcl-Associated Death Protein
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