NGF protects PC12 cells against ischemia by a mechanism that requires the N-kinase.

Nerve growth factor (NGF), which has been shown to act as a morphological and neurochemical differentiating factor in PC12 cells, also protects PC12 cells from the toxicity of serum withdrawal and ischemia. By using a previously established in vitro model of ischemia, which incorporates the combination of anoxia with glucose deprivation (Boniece and Wagner: J Neurosci 13:4220-4228, 1993), we have been able to study the signal transduction pathways upon which NGF-induced survival is dependent. Here we demonstrate that inhibitors of the N-kinase and NGF-induced neuritogenesis, 6-thioguanine and 2-aminopurine, prevent the protective effects of NGF, while they have little, if any, effect on the protection conferred by epidermal growth factor (EGF) or dbcAMP. This suggests that only NGF acts by a mechanism that depends strongly on the N-kinase. Furthermore, the methyltransferase inhibitor 5'-deoxy-5'-methylthioadenosine (MTA), which also inhibits NGF-induced neuritogenesis, inhibits the protective effect of NGF but not the protective effects of EGF or dbcAMP. Thus, the neuroprotective effect of NGF requires some of the same signal transduction steps used by NGF to promote differentiation and neurite formation. Furthermore, we found that exposure of PC12 cells to retinoic acid, which promotes the differentiation and inhibits the growth of PC12 cells, also improves cell survival during ischemia. In addition, a combination of NGF and retinoic acid was more effective than either agent alone. It is likely that these two agents confer protection by independent pathways.

Growth factors protect PC12 cells against ischemia by a mechanism that is independent of PKA, PKC, and protein synthesis.

We have established an in vitro model of ischemia incorporating the combination of anoxia with glucose deprivation, which is toxic to PC12 cells. In this model, nerve growth factor (NGF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) improve PC12 cell survival. K252a, a specific inhibitor of NGF-induced trk p140 autophosphorylation, did not alter the neuroprotection provided by EGF or bFGF, yet it completely abolished the protection provided by NGF. Activation of protein kinase A (PKA) with dibutyryl-cAMP also protected during ischemia, although it was not additive with the effect provided by growth factors. Furthermore, growth factors protected a PKA-deficient mutant as effectively as the parental cell line; thus, activation of PKA is protective against ischemia but is not necessary for the action of peptide growth factors. Neither the stimulation of protein kinase C (PKC) with acute phorbol ester treatment nor the downregulation of PKC with chronic high-dose phorbol ester treatment resulted in an altered response to growth factors in either the PC12 wild type or PKA-deficient mutant. Thus, protection by peptide growth factors depends on neither PKA nor PKC. Furthermore, downregulation of PKC alone was protective, indicating that PKC may contribute to toxicity. Interestingly, treatment with the kinase inhibitor H-7 was neuroprotective and may have enhanced the neuroprotective effect of NGF. In contrast, staurosporine, a broadly acting kinase inhibitor, inhibited the neuroprotective effect of NGF, but not of EGF or FGF.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein kinases modulate the sensitivity of hippocampal neurons to nitric oxide toxicity and anoxia.

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.

Peptide growth factors protect against ischemia in culture by preventing nitric oxide toxicity.

Reduction or elimination of nitric oxide (NO) production in cortical neurons by NO synthase (NOS) inhibitors during glutamate toxicity in vitro or during focal cerebral ischemia in vivo can prevent neuronal cell death. In contrast, growth factors can prevent neuronal degeneration induced by treatment with glutamate or potassium cyanide. We have determined whether NO mediates hippocampal cell death during anoxia in vitro and whether the peptide growth factors basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) can prevent hippocampal neuronal death during anoxia or NO exposure. Both bFGF and EGF increased hippocampal neuronal survival from about 35% in anoxic cultures to about 65% in treated cultures during an 8 hr period of anoxia. Inhibition of NOS by NG-monomethyl-L-arginine, a competitive inhibitor of NOS, rescued 65-70% of the neurons that would normally die during an 8 hr anoxic incubation, and this effect was reversed by L-arginine, a precursor for NO. Thus, hippocampal neuronal death following anoxia is, at least in part, mediated by NO. NO, generated by either nitroprusside or 3-morpholino-sydnonimine, was toxic to hippocampal neurons. Pretreatment of cultures with either bFGF (10 ng/ml) or EGF (10 ng/ml) prior to NO exposure increased survival from approximately 40% in untreated cultures to 80% in treated cultures, yet the effect of combining bFGF and EGF was not greater than treatment with either of the growth factors alone. Knowledge that the growth factors bFGF and EGF are neuroprotective against NO toxicity provides insights into the mechanisms of ischemic neuronal death that may direct future therapeutic modalities for cerebrovascular disease and neurodegenerative disorders.