DOWN-REGULATION OF NADPH-DIAPHORASE (NITRIC-OXIDE SYNTHASE) MAY ACCOUNT FOR THE PHARMACOLOGICAL ACTIVITIES OF CU(II)(2)(3,5-DIISOPROPYLSALICYLATE)(4)

Purposes of this work were to develop an enzyme system as an in vitro model of the NADPH-dependent component of nitric oxide synthase (NOS) and examine the plausible down-regulation of this system and brain NOS by copper (II)(2)(3,5-diisopropylsalicylate)(4)[Cu(II)(2)(3,5-DIPS)(4)] as a mechanism accounting for its analgesic, anticonvulsant, and other pharmacological activities. Porcine heart diaphorase (PHD) was found to oxidize 114 mu M NADPH with the corrsponding reduction of an equivalent amount of 2,6-dichlorophenolindophenol (DCPIP). Addition of Cu(II)(2)(3,5-DIPS)(4) to the reaction mixture decreased the reduction of DCPIP without substantially affecting the oxidation of NADPH. The IC50 for Cu(II)(2)(3,5-DIPS)(4) in inhibiting the reduction of DCPIP was 1.5 mu M. Mechanistically, this inhibition of DCPIP reduction was found to be due to the ability of Cu(II)(2)(3,5-DIPS)(4) to serve as a catalytic electron acceptor for reduced PHD, which was enhanced by the presence of a large concentration of DCPIP and inhibited by a large concentration of NADPH. Oxidation of NADPH by PHD in the absence of DCPIP was linearly related to the concentration of Cu(II)(2)(3,5-DIPS)(4) through the concentration range of 5-25 mu M Cu(II)(2)(3,5-DIPS)(4) with 50% recovery of NADPH oxidation by PHD at a concentration of 16 mu M Cu(II)(2)(3,5-DIPS)(4). Whole rat brain tissue sections incubated in medium containing an NADPH-generating system and nitroblue tetrazolium chloride (NBT) were less intensely stained when Cu(II)(2)(3,5-DIPS)(4) was added to the medium. It is concluded that Cu(II)(2)(3,5-DIPS)(4) serves as an electron acceptor in down-regulating PHD reduction of DCPIP and in down-regulating NOS in brain tissue sections. A decrease in NO synthesis in animal models of seizure, pain, and other disease states with Cu(II)(2)(3,5-DIPS)(4) may account for the anticonvulsant, analgesic, and other pharmacological activities of this complex.