Metabotropic glutamate receptors prevent programmed cell death through the modulation of neuronal endonuclease activity and intracellular pH

Abstract

Metabotropic glutamate receptor (mGluR) activation prevents neurodegeneration against nitric oxide (NO)-induced programmed cell death (PCD). We therefore investigated whether specific neuronal endogenous deoxyribonucleases, enzymes recently identified to be responsible for the maintenance of DNA integrity, mediated mGluR protection against NO. In rat primary hippocampal neurons, injury was assessed by using a 0.4% trypan blue dye exclusion method and TUNEL assay 24 h following treatment with the NO generators sodium nitroprusside (300 microM) or SIN-1 (300 microM). DNA digestion studies using neuronal cell extracts were employed to assess specific endonuclease activity. Individual application of aurintricarboxylic acid (ATA) (10 microM), an endonuclease inhibitor, or the mGluR agonists 1S,3R-ACPD (750 microM), DHPG (750 microM), L-CCG-I (750 microM), or L-AP4 (750 microM) prior to NO exposure significantly increased neuronal survival. Yet, combination therapy with ATA (10 microM) and the mGluR agonists did not synergistically improve neuronal survival, suggesting a common pathway of protection for ATA and the mGluRs that is dependent upon the modulation of neuronal endonuclease activity. In further support of this premise, protection by the mGluR agonists 1S,3R-ACPD, DHPG, L-CCG-I, and L-AP4 was significantly decreased during enhancement of endonuclease activity with the zinc chelator, N,N,N',N',-tetrakis (2-pyridylmethyl) ethylenediamine. Antagonism of the mGluR system was ineffective against endonuclease induced DNA destruction. Further assessment with DNA digestion assays identified two distinct mechanisms to maintain DNA integrity, a Ca2+/Mg2+-dependent endonuclease inhibited by L-AP4 and a magnesium dependent endonuclease inhibited by 1S,3R-ACPD. These neuroprotective mechanisms during activation of the mGluR system were also intricately linked to the active reversal of the biphasic intracellular pH changes induced by NO. Further investigation into the molecular pathways modulated by mGluRs may identify specific mechanisms that can maintain DNA integrity during adverse cellular environments.