title: Inhibition of Bax-induced cytochrome c release from neural cell and brain mitochondria by dibucaine and propranolol.
abstract: BH3 (Bcl-2 homology 3)-only proteins of the Bcl-2 family activate Bax or Bak during apoptosis to promote the release of pro-death factors sequestered in the mitochondrial intermembrane space. Previous results demonstrated that a synthetic BH3 peptide mimics the ability of the BH3-only protein Bid to promote Bax insertion and cytochrome c (cyt c) release from neural cell mitochondria. However, the BH3 peptide was deficient in promoting cyt c release from mitochondria without associated Bax, such as adult rat brain mitochondria. This study tested the hypothesis that the amphiphilic membrane-active cationic drugs dibucaine and propranolol block BH3 peptide-initiated cyt c efflux by preventing the integration of Bax into the mitochondrial outer membrane. BH3 peptide-initiated release of cyt c from GT1-7 neural cell mitochondria was inhibited by dibucaine and propranolol at concentrations of 100-300 microm. Recombinant Bax (100 nm) alone did not release cyt c from adult rat brain mitochondria; however, when BH3 peptide or caspase-8 cleaved Bid (cBid) was added, robust cyt c release was achieved that was inhibited completely by 200 microm dibucaine or propranolol. These drugs at similar concentrations also inhibited release of entrapped 10 kDa dextrans from protein-free liposomes treated with Bax and cBid. Contrary to the hypothesis that dibucaine and propranolol act by inhibiting the insertion of Bax into the mitochondrial outer membrane, membrane insertion of Bax was not inhibited in mitochondria or liposomes, indicating a mechanism of drug action downstream from this event. These results suggest that dibucaine and propranolol inhibit Bax-induced permeability changes through a direct interaction with the lipid membrane and present a novel target for the development of neuroprotective, antiapoptotic therapeutics.
authors.affiliation: Department of Anesthesiology and Neuroscience Program, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
authors.forename: Brian M
authors.initials: BM
authors.lastname: Polster
authors.forename: Gorka
authors.initials: G
authors.lastname: Basañez
authors.forename: Michael
authors.initials: M
authors.lastname: Young
authors.forename: Motoshi
authors.initials: M
authors.lastname: Suzuki
authors.forename: Gary
authors.initials: G
authors.lastname: Fiskum
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D051023:BH3 Interacting Domain Death Agonist Protein; D001692:Biological Transport; D001921:Brain / Q000378:metabolism / Q000648:ultrastructure; D002352:Carrier Proteins / Q000378:metabolism; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D003574:Cytochrome c Group / Q000378:metabolism*; D003911:Dextrans / Q000378:metabolism; D003992:Dibucaine / Q000494:pharmacology*; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D007473:Ion Channels / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D008969:Molecular Sequence Data; D009474:Neurons / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D016716:PC12 Cells; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000737:chemistry; D011433:Propranolol / Q000494:pharmacology*; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2; D051381:Rats; D017207:Rats, Sprague-Dawley; D051028:bcl-2-Associated X Protein
chemical_list: C494826:BAX protein, human; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; C474007:Bax protein (53-86); C494828:Bax protein, rat; C494816:Bid protein, rat; D002352:Carrier Proteins; D003574:Cytochrome c Group; D003911:Dextrans; D007473:Ion Channels; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D011433:Propranolol; C505530:CASP8 protein, human; C506433:CASP9 protein, human; C505528:Casp8 protein, rat; C506435:Casp9 protein, rat; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D003992:Dibucaine