Multiple processes lead to neuronal death after
ischemia, but the generation of
nitric oxide (NO) is a key component in this cascade of events. The mechanisms that regulate the extent of neuronal degeneration during
anoxia and NO toxicity are multifactorial. Neuronal death may be modulated by the activity of signal transduction systems that influence the toxicity of NO or its metabolic products such as cGMP. The
enzyme responsible for the production of NO,
nitric oxide synthase (NOS), is phosphorylated by
protein kinase C (PKC), the
cAMP-dependent protein kinase (PKA), and the
calcium/calmodulin-dependent protein kinase II (CaM-II). We examined in primary cultured hippocampal neurons whether the
protein kinases PKC, PKA, CaM-II, and
cGMP-dependent protein kinase modified the toxic effects of
anoxia and NO. Down-regulation of PKC activity with PMA (1 microM) increased hippocampal neuronal survival during
anoxia and NO exposure from approximately 22% to 88%. Inhibitors of PKC activity (
H-7, H-8,
sphingosine, and
staurosporine) also were neuroprotective. Down-regulation of PKC activity increased survival during
anoxia even in the presence of the NOS inhibitor, N omega-methyl-
L-arginine. Thus, although down-regulation of PKC activity may increase neuronal survival by decreasing NOS activity, it also is likely that PKC contributes to ischemic neuronal death by mechanisms that are independent of NOS. Inhibition of the
cGMP-dependent protein kinase activity, but not the activity of the CaM-II also was neuroprotective during NO administration. In contrast to the protective effects of inhibition of PKC and the
cGMP-dependent protein kinase, activation rather than inhibition of PKA increased hippocampal neuronal survival during NO exposure. These results indicate that neuronal survival during
anoxia and NO exposure is linked to the modulation of PKC, PKA, and
cGMP-dependent protein kinase activity but is not dependent on the CaM-II pathway. Understanding the involvement of PKC, PKA, and the
cGMP-dependent protein kinase in modulating the effect of neuronal death during
ischemia and NO toxicity may help in directing future therapeutic modalities for
cerebrovascular disease.