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The neuroprotective effect of MK801 against hypoxia and/or reoxygenation-induced neuronal cell injury and its relationship to neuronal nitric oxide synthetase (nNOS) expression were examined in cultured rat cortical cells. Treatment of cortical neuronal cells with hypoxia (95% N(2)/5% CO(2)) for 2 h followed by reoxygenation for 24 h induced a release of lactate dehydrogenase (LDH) into the medium, and reduced the protein level of MAP-2 as well. MK801 attenuated the release of LDH and the reduction of the MAP-2 protein by hypoxia, suggesting a neuroprotective role of MK801. MK801 also diminished the number of nuclear condensation by hypoxia/reoxygenation. The NOS inhibitors 7-nitroindazole (7-NI) and N (G)-nitro-L-arginine methyl ester (L-NAME), as well as the Ca(2+) channel blocker nimodipine, reduced hypoxia-induced LDH, suggesting that nitric oxide (NO) and calcium homeostasis contribute to hypoxia and/or the reoxygenation-induced cell injury. The levels of nNOS immunoactivities and mRNA by RT-PCR were enhanced by hypoxia with time and, down regulated following 24 h reoxygenation after hypoxia, and were attenuated by MK801. In addition, the reduction of nNOS mRNA levels by hypoxia/reoxygenation was also diminished by MK801. Further delineation of the mechanisms of NO production and nNOS regulation are needed and may lead to additional strategies to protect neuronal cells against hypoxic/reoxygenation insults.

Signal transduction systems, including cholinergic pathways, which are likely to be of pathophysiological significance are altered in Alzheimer's disease (AD). Muscarinic cholinergic receptors are linked to the hydrolysis of phosphoinositide, involving the production of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and the mobilization of cytosolic free calcium concentrations ([Ca2+]i). Effects of amyloid peptide (A(beta)) on these signals prior to neuronal degeneration were examined in cultured rat cortical cells. A(beta) increased the release of lactate dehydrogenase (LDH) in a concentration-dependent manner, however, it was blocked by B27 supplement. Prolonged exposure to a sublethal dose of A(beta) 25-35 or 1-42 disrupted carbachol-mediated release of Ins(1,4,5)P3 and [Ca2+]i, which was inhibited in media supplemented with B27 or the antioxidant vitamin E. In order to determine the specificity of the effect of A(beta), various agonists glutamate or KCl but not bradykinin which utilize the phosphoinositide cascade were investigated. Our results indicated that A(beta) did not affect the stimulation of glutamate or KCl-mediated production of Ins(1,4,5)P3 or cause elevation in [Ca2+]i. Furthermore, metabotropic agonist trans-1-amino-cyclopentane-1,3,-dicarboxylate (ACPD) elevated calcium level was not inhibited by A(beta) pre-treatment. Taken together, the results demonstrate that a sublethal dose of A(beta) selectively impaired cholinergic receptor-mediated signal transduction pathways, and antioxidant or B27 supplement attenuated this effect of A(beta). Alterations of cholinergic signaling by prolonged exposure to A(beta) could be involved in cortical neurodegeneration that occurs in AD. Because functional loss of cholinergic pathways is an important aspect of AD, the differences in susceptibility of these two types of receptors prior to other signs of A(beta) action is important and requires further investigation.