Carbon monoxide abrogates ischemic insult to neuronal cells via the soluble guanylate cyclase-cGMP pathway

PLoS One. 2013 Apr 8;8(4):e60672. doi: 10.1371/journal.pone.0060672. Print 2013.

Abstract

Purpose: 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis / drug effects
  • Brain Ischemia / enzymology
  • Brain Ischemia / metabolism
  • Brain Ischemia / pathology*
  • Brain Ischemia / prevention & control
  • Carbon Monoxide / metabolism*
  • Carbon Monoxide / pharmacology
  • Cell Line, Tumor
  • Coordination Complexes / chemistry
  • Coordination Complexes / metabolism
  • Coordination Complexes / pharmacology
  • Cyclic GMP / metabolism*
  • Dose-Response Relationship, Drug
  • Female
  • Gene Expression Regulation, Enzymologic / drug effects
  • Guanylate Cyclase / genetics
  • Guanylate Cyclase / metabolism*
  • Humans
  • Male
  • Molybdenum / chemistry
  • NADPH Oxidases / metabolism
  • Neurons / drug effects
  • Neurons / enzymology
  • Neurons / metabolism*
  • Neuroprotective Agents / metabolism*
  • Neuroprotective Agents / pharmacology
  • Organometallic Compounds / chemistry
  • Organometallic Compounds / metabolism
  • Organometallic Compounds / pharmacology
  • Rats
  • Rats, Sprague-Dawley
  • Reactive Oxygen Species / metabolism
  • Receptors, Cytoplasmic and Nuclear / genetics
  • Receptors, Cytoplasmic and Nuclear / metabolism*
  • Reperfusion Injury / enzymology
  • Reperfusion Injury / prevention & control
  • Retinal Ganglion Cells / drug effects
  • Retinal Ganglion Cells / metabolism
  • Retinal Ganglion Cells / pathology
  • Rotenone / toxicity
  • Signal Transduction / drug effects*
  • Soluble Guanylyl Cyclase

Substances

  • Coordination Complexes
  • Neuroprotective Agents
  • Organometallic Compounds
  • Reactive Oxygen Species
  • Receptors, Cytoplasmic and Nuclear
  • triscarbonyl(histidinato)molybdate(III)
  • Rotenone
  • Carbon Monoxide
  • Molybdenum
  • NADPH Oxidases
  • Guanylate Cyclase
  • Soluble Guanylyl Cyclase
  • Cyclic GMP