title: Cytosolic Ca2+-induced apoptosis in rat cardiomyocytes via mitochondrial NO-cGMP-protein kinase G pathway.
abstract: Previously, we showed that in adult rat cardiomyocytes, nitric oxide (NO) donors stimulate mitochondrial cGMP production, followed by cytochrome c release, independently of the mitochondrial permeable transition pore. We investigated whether mitochondrial cGMP-induced cytochrome c release from cardiac mitochondria is Ca(2+)-sensitive. Mitochondria and primary cultured cardiomyocytes were prepared from left ventricles of male Wistar rats. The cytosolic Ca(2+) concentration was adjusted with Ca(2+)-EGTA buffers. Cytochrome c released from mitochondria was measured by Western blotting. Cardiomyocyte apoptosis was assessed by Annexin V staining. Cytochrome c release from cardiac mitochondria was evoked by buffered Ca(2+) (1 μM); this was inhibited by NO-cGMP pathway inhibitors such as N(G)-monomethyl-l-arginine monoacetate (inhibitor of NO synthase), 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (NO scavenger), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, NO-sensitive guanylyl cyclase inhibitor) and voltage-dependent anion channel (VDAC) inhibitor, 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene, but not by cyclosporin A (mitochondrial permeable transition pore inhibitor). Furthermore, this release was significantly and dose dependently inhibited by 0.3-3 μM KT5823 (protein kinase G inhibitor). At the cellular level, intracellular perfusion of cardiomyocytes with buffered Ca(2+) (1 μM) also induced apoptosis, which was inhibited in the presence of ODQ. A membrane-permeable cGMP analog, 8-Br-cGMP, but not cGMP itself, mimicked buffered Ca(2+) actions in both cardiac mitochondria and cardiomyocytes. We further confirmed an increase in protein kinase G activity by adding cGMP in mitochondrial protein fraction. Our results suggest that mitochondrial NO-cGMP pathway-induced cytochrome c release from cardiac mitochondria, triggered by increased cytosolic Ca(2+), occurs through VDAC via the stimulation of an undiscovered mitochondrial protein kinase G.
authors.affiliation: Department of Pharmacology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan.
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Seya
authors.forename: Kyoichi
authors.initials: K
authors.lastname: Ono
authors.forename: Susumu
authors.initials: S
authors.lastname: Fujisawa
authors.forename: Ken
authors.initials: K
authors.lastname: Okumura
authors.forename: Shigeru
authors.initials: S
authors.lastname: Motomura
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Furukawa
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000494:pharmacology*; D020013:Calcium Signaling / Q000187:drug effects; D002469:Cell Separation; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D003600:Cytosol / Q000502:physiology*; D016166:Free Radical Scavengers; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008929:Mitochondria, Heart / Q000187:drug effects*; D008933:Mitochondrial Swelling / Q000187:drug effects; D032383:Myocytes, Cardiac / Q000187:drug effects*; D009569:Nitric Oxide / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D018408:Patch-Clamp Techniques; D051381:Rats; D017208:Rats, Wistar
chemical_list: D016166:Free Radical Scavengers; C016276:8-bromocyclic GMP; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D017869:Cyclic GMP-Dependent Protein Kinases; D006152:Cyclic GMP; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D002118:Calcium