title: Carbon monoxide abrogates ischemic insult to neuronal cells via the soluble guanylate cyclase-cGMP pathway.
abstract: OBJECTIVE
  Carbon monoxide (CO) is an accepted cytoprotective molecule. The extent and mechanisms of protection in neuronal systems have not been well studied. We hypothesized that delivery of CO via a novel releasing molecule (CORM) would impart neuroprotection in vivo against ischemia-reperfusion injury (IRI)-induced apoptosis of retinal ganglion cells (RGC) and in vitro of neuronal SH-SY5Y-cells via activation of soluble guanylate-cyclase (sGC).
  
  
  METHODS
  To mimic ischemic respiratory arrest, SH-SY5Y-cells were incubated with rotenone (100 nmol/L, 4 h) ± CORM ALF186 (10-100 µmol/L) or inactivated ALF186 lacking the potential of releasing CO. Apoptosis and reactive oxygen species (ROS) production were analyzed using flow-cytometry (Annexin V, mitochondrial membrane potential, CM-H2DCFDA) and Western blot (Caspase-3). The impact of ALF186± respiratory arrest on cell signaling was assessed by measuring expression of nitric oxide synthase (NOS) and soluble guanylate-cyclase (sGC) and by analyzing cellular cGMP levels. The effect of ALF186 (10 mg/kg iv) on retinal IRI in Sprague-Dawley rats was assessed by measuring densities of fluorogold-labeled RGC after IRI and by analysis of apoptosis-related genes in retinal tissue.
  
  
  RESULTS
  ALF186 but not inactivated ALF186 inhibited rotenone-induced apoptosis (Annexin V positive cells: 25 ± 2% rotenone vs. 14 ± 1% ALF186+rotenone, p<0.001; relative mitochondrial membrane potential: 17 ± 4% rotenone vs. 55 ± 3% ALF186+rotenone, p<0.05). ALF186 increased cellular cGMP levels (33±5 nmol/L vs. 23±3 nmol/L; p<0.05) and sGC expression. sGC-inhibition attenuated ALF186-mediated protection (relative mitochondrial membrane potential: 55±3% ALF186+rotenone vs. 20 ± 1% ODQ + ALF186+rotenone, p<0.05). ALF186 protected RGC in vivo (IRI 1255 ± 327 RGC/mm(2) vs. ALF186 + IRI 2036 ± 83; p<0.05) while sGC inhibition abolished the protective effects of ALF186 (ALF186 + IRI 2036 ± 83 RGC/mm(2) vs. NS-2028 + ALF186 + IRI 1263 ± 170, p<0.05).
  
  
  CONCLUSIONS
  The CORM ALF186 inhibits IRI-induced neuronal cell death via activation of sGC and may be a useful treatment option for acute ischemic insults to the retina and the brain.
authors.affiliation: Department of Anesthesiology, Division for Experimental Anesthesiology, University Medical Center Freiburg, Germany. nils.schallner@uniklinik-freiburg.de
authors.forename: Nils
authors.initials: N
authors.lastname: Schallner
authors.forename: Carlos C
authors.initials: CC
authors.lastname: Romão
authors.forename: Julia
authors.initials: J
authors.lastname: Biermann
authors.forename: Wolf A
authors.initials: WA
authors.lastname: Lagrèze
authors.forename: Leo E
authors.initials: LE
authors.lastname: Otterbein
authors.forename: Hartmut
authors.initials: H
authors.lastname: Buerkle
authors.forename: Torsten
authors.initials: T
authors.lastname: Loop
authors.forename: Ulrich
authors.initials: U
authors.lastname: Goebel
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002545:Brain Ischemia / Q000201:enzymology / Q000378:metabolism / Q000473:pathology* / Q000517:prevention & control; D002248:Carbon Monoxide / Q000378:metabolism* / Q000494:pharmacology; D045744:Cell Line, Tumor; D056831:Coordination Complexes / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D006152:Cyclic GMP / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005260:Female; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006162:Guanylate Cyclase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008297:Male; D008982:Molybdenum / Q000737:chemistry; D019255:NADPH Oxidases / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D018696:Neuroprotective Agents / Q000378:metabolism* / Q000494:pharmacology; D009942:Organometallic Compounds / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D015427:Reperfusion Injury / Q000201:enzymology / Q000517:prevention & control; D012165:Retinal Ganglion Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D012402:Rotenone / Q000633:toxicity; D015398:Signal Transduction / Q000187:drug effects*; D000071756:Soluble Guanylyl Cyclase
chemical_list: D056831:Coordination Complexes; D018696:Neuroprotective Agents; D009942:Organometallic Compounds; D017382:Reactive Oxygen Species; D018160:Receptors, Cytoplasmic and Nuclear; C000620213:triscarbonyl(histidinato)molybdate(III); D012402:Rotenone; D002248:Carbon Monoxide; D008982:Molybdenum; D019255:NADPH Oxidases; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase; D006152:Cyclic GMP