Role of cyclic GMP in glutamate neurotoxicity in primary cultures of cerebellar neurons

Abstract

The role of cGMP in the mediation of glutamate neurotoxicity remains controversial. Some reports indicate that cGMP mediates glutamate neurotoxicity while others indicate that cGMP is neuroprotective. We have studied the role of cGMP in the mediation of glutamate and nitric oxide neurotoxicity in primary cultures of cerebellar neurons. Inhibition of soluble guanylate cyclase prevents glutamate and nitric oxide neurotoxicity. There is a good correlation between inhibition of cGMP formation and neuroprotection. Moreover 8-Br-cGMP, a cell permeable analog of cGMP, induced neuronal death. These results indicate that increased intracellular cGMP is involved in the mechanism of neurotoxicity. Inhibitors of phosphodiesterase did not increase intracellular cGMP but increased the content of cGMP in the extracellular medium and prevented glutamate neurotoxicity. Moreover, addition of cGMP to the extracellular medium also prevented glutamate neurotoxicity in cerebellar neurons in culture. These results are compatible with a neurotoxic effect of increased intracellular cGMP and a neuroprotective effect of increased extracellular cGMP.

Introduction

Glutamate is the main excitatory neurotransmitter in mammals. However, excessive activation of glutamate receptors leads to neuronal degeneration and death. Excitatory amino acid neurotoxicity has been proposed to contribute to the pathogenesis of different neurodegenerative diseases, including amyotrophic lateral sclerosis and Huntington disease. Glutamate neurotoxicity is also involved in the neuronal damage found in cerebral ischemia and it has been proposed to be involved in the origin of Alzheimer’s and Parkinson’s diseases. The understanding of the molecular mechanism of glutamate neurotoxicity and of the possible mechanisms to prevent it would be therefore of great interest for the treatment of the above situations.

In many systems, including primary cultures of cerebellar neurons, glutamate neurotoxicity is mainly mediated by excessive activation of the NMDA type of glutamate receptor (Choi, 1987, Novelli et al., 1988, Miñana et al., 1996). Increased intracellular Ca²⁺ is an essential step leading to neuronal death (Choi, 1987, Manev et al., 1989). In several types of neurons in culture one step in the neurotoxic process is the glutamate-induced formation of nitric oxide. This is supported by experiments showing that glutamate neurotoxicity is prevented by inhibitors of nitric oxide synthase such as nitroarginine (Dawson et al., 1991, Dawson et al., 1993, Cazevieille et al., 1993, Lafon-Cazal et al., 1993). Moreover, it has been proposed that prolonged application of nitroarginine enhances the neuroprotective efficacy (Vigé et al., 1993). In the cerebellar neurons used in the present work, glutamate neurotoxicity is completely prevented by nitroarginine (Marcaida et al., 1995). It seems therefore that formation of nitric oxide is an essential step in the mediation of glutamate neurotoxicity. However, the subsequent events in the neurotoxic process are not well understood.

Nitric oxide activates soluble guanylate cyclase and increases the cGMP concentration. Activation of ionotropic receptors leads to activation of nitric oxide synthase, increased formation of nitric oxide and an increased concentration of cGMP (Bredt and Snyder, 1989, East and Garthwaite, 1990, Southam et al., 1991). However, the role of cGMP in the mediation of glutamate neurotoxicity remains controversial. Some authors indicate that cGMP plays a role in the mechanism of glutamate neurotoxicity; however, other authors suggest that cGMP could have a neuroprotective effect (see Section 4).

We have recently shown that 1H-[1,2,4]oxadiazol-[4,3a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase prevents glutamate neurotoxicity (Hermenegildo et al., 1998). In the present work we have studied in more detail the role of glutamate-induced formation of cGMP in the mechanism of glutamate neurotoxicity and its possible neuroprotective effect. ODQ, an inhibitor of soluble guanylate cyclase prevents glutamate neurotoxicity. Three different inhibitors of phosphodiesterase (3-isobutyl-1-methylxanthine, zaprinast and nicardipine) also prevent glutamate neurotoxicity. The results reported suggest that intracellular accumulation of cGMP is necessary but not sufficient for the mediation of glutamate neurotoxicity and that extracellular cGMP can afford protection against glutamate neurotoxicity.