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

Grant support

This work was funded by departmental funding of the Department of Anesthesiology and Critical Care Medicine and the University Eye Hospital, University Medical Center Freiburg, Germany. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.