Elevated endogenous nitric oxide increases Ca2+ flux via L-type Ca2+ channels by S-nitrosylation in rat hippocampal neurons during severe hypoxia and in vitro ischemia

Free Radic Biol Med. 2007 Jan 1;42(1):52-63. doi: 10.1016/j.freeradbiomed.2006.09.020. Epub 2006 Sep 27.


Nitric oxide (NO) mediates pathogenic changes in the brain subsequent to energy deprivation; yet the NO mechanism involved in the early events remains unclear. We examined the acute effects of severe hypoxia and oxygen-glucose deprivation (OGD) on the endogenous NO production and the NO-mediated pathways involved in the intracellular calcium ([Ca(2+)](i)) response in the rat hippocampal neurons. The levels of NO and [Ca(2+)](i) in the CA1 region of the slices rapidly elevated in hypoxia and were more prominent in OGD, measured by the electrochemical method and spectrofluorometry, respectively. The NO and [Ca(2+)](i) responses were enhanced by L-arginine and were reduced by NO synthase inhibitors, suggesting that the endogenous NO increases the [Ca(2+)](i) response to energy deprivation. Nickel and nifedipine significantly decreased the NO and [Ca(2+)](i) responses to hypoxia and OGD, indicating an involvement of L-type Ca(2+) channels in the NO-mediated mechanisms. In addition, the [Ca(2+)](i) responses were attenuated by ODQ or KT5823, inhibitors of the cGMP-PKG pathway, and by acivicin, an inhibitor of gamma-glutamyl transpeptidase for S-nitrosylation, and by the thiol-alkylating agent N-ethylmaleimide (NEM). Moreover, L-type Ca(2+) currents in cultured hippocampal neurons with whole-cell recording were significantly increased by L-arginine and were decreased by L-NAME. Pretreatment with NO synthase inhibitors or NEM but not ODQ abolished the effect of L-arginine on the Ca(2+) currents. Also, vitamin C, which decomposes nitrosothiol but not disulfide by reduction, reversed the change in the Ca(2+) current with L-arginine. Taken together, the results suggest that an elevated endogenous NO production enhances the influx of Ca(2+) via the hippocampal L-type Ca(2+) channel by S-nitrosylation during an initial phase of energy deprivation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Arginine / pharmacology
  • Brain Ischemia / metabolism*
  • Brain Ischemia / pathology
  • Calcium / metabolism*
  • Calcium Channels, L-Type / metabolism*
  • Cell Hypoxia*
  • Cells, Cultured
  • Cyclic GMP / metabolism
  • Electrophysiology
  • Enzyme Inhibitors / pharmacology
  • Fura-2
  • Hippocampus / cytology
  • Hippocampus / metabolism*
  • NG-Nitroarginine Methyl Ester / pharmacology
  • Neurons / cytology
  • Neurons / metabolism*
  • Nitric Oxide / pharmacology*
  • Nitric Oxide Synthase / antagonists & inhibitors
  • Nitric Oxide Synthase / metabolism
  • Oxygen / metabolism
  • Rats
  • Rats, Sprague-Dawley
  • S-Nitrosothiols / metabolism


  • Calcium Channels, L-Type
  • Enzyme Inhibitors
  • S-Nitrosothiols
  • Nitric Oxide
  • Arginine
  • Nitric Oxide Synthase
  • Cyclic GMP
  • Oxygen
  • Calcium
  • Fura-2
  • NG-Nitroarginine Methyl Ester