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 microM NADPH with the corresponding 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 microM. 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 microM Cu(II)2(3,5-DIPS)4 with 50% recovery of NADPH oxidation by PHD at a concentration of 16 microM 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.
Department of Medicinal Chemistry, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock 72205, USA.
J G
JG
Baquial
J R
JR
Sorenson
D000818:Animals; D001391:Azo Compounds / Q000378:metabolism; D001921:Brain / Q000201:enzymology*; D015536:Down-Regulation; D004579:Electron Transport; D007004:Hypoglycemic Agents / Q000494:pharmacology; D015394:Molecular Structure; D009206:Myocardium / Q000201:enzymology; D009249:NADP / Q000378:metabolism; D009252:NADPH Dehydrogenase / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D010084:Oxidation-Reduction; D012459:Salicylates / Q000494:pharmacology*; D013053:Spectrophotometry; D013552:Swine
D001391:Azo Compounds; D007004:Hypoglycemic Agents; D012459:Salicylates; C033302:diformazan dye; C030865:copper bis(3,5-diisopropylsalicylate); D009249:NADP; D019001:Nitric Oxide Synthase; D009252:NADPH Dehydrogenase