CYANIDE-INDUCED GENERATION OF OXIDATIVE SPECIES - INVOLVEMENT OF NITRIC-OXIDE SYNTHASE AND CYCLOOXYGENASE-2

In cerebellar granule cells, potassium cyanide (KCN) activates the NMD A receptor resulting in generation of nitric oxide and reactive oxygen species (ROS). To study the mechanism by which KCN stimulates ROS gen eration, the action of cyanide on the enzymatic pathways known to gene rate ROS were studied. The oxidant-sensitive fluorescent dye, 2,7-dich lorofluorescin was used to measure intracellular levels of nitric oxid e and ROS in cerebellar granule cells. Using selective enzyme inhibito rs, it was shown that both protein kinase C and phospholipase A(2) are involved in KCN-stimulated generation of NO and ROS. In cells treated with indomethacin or nordihydroguairetic acid, inhibitors of cyclooxy genase (COX) and lipoxygenase (LOX) respectively, attenuated (similar to 35%) KCN-induced generation of oxidant species. When L-NAME (L-G-ni tro-L-arginine methyl ester) (nitric oxide synthase inhibitor, NOS) wa s combined with either indomethacin or nordihydroguairetic acid, gener ation of oxidant species was blocked by more than 80%. Pretreatment wi th NS398 (COX-2 inhibitor) significantly decreased ROS generation indi cating the involvement of COX-2 in KCN-induced oxidant generation. Tre atment with L-NAME + NS398 blocked oxidant species generation, reflect ing involvement of NOS. The participation of cytochrome P450 was not e vident because SKF525A did not significantly reduce KCN-induced ROS ge neration. Furthermore, a correlation was observed between oxidant gene ration and lipid peroxidation of cellular membranes (as determined by thiobarbituric acid levels). Pretreatment with inhibitors of protein k inase C, phospholipase A(2) or COX, LOX, COX-2 partially blocked KCN-i nduced formation of thiobarbituric acid reactive substance, whereas co incubation of L-NAME with the inhibitors decreased lipid peroxidation by 60 to 90%. In cytotoxicity studies, KCN-induced cell death was part ially blocked by the inhibitors and significant protection was observe d when L-NAME was combined with these compounds. These findings show t hat activation of phospholipase A(2) and subsequent metabolism of arac hidonic acid by the COX-2 and LOX pathways and NOS contribute to cyani de-induced ROS production.