Down‐Regulation of Porcine Heart Diaphorase Reactivity byTrimanganese Hexakis(3,5‐Diisopropylsalicylate),Mn3(3,5‐DIPS) 6 , andDown‐Regulation of Nitric Oxide Synthase Reactivity by Mn3(3,5‐DIPS) 6 and Cu(II) 2(3,5‐DIPS) 4

Abstract

Purposes of this work were to examine the plausible down-regulation of porcine heart diaphorase (PHD) enzyme reactivity and nitric oxide synthase (NOS) enzyme reactivity by trimanganese hexakis(3,5-diisopropylsalicylate), [Mn3(3,5-DIPS)6] as well as dicopper tetrakis(3,5- diisopropylsalicylate, [Cu(II)2(3,5-DIPS)4] as a mechanistic accounting for their pharmacological activities. Porcine heart disease was found to oxidize 114 μM reduced nicotinamide-adenine- dinucleotide-′3-phosphate (NADPH) with a corresponding reduction of an equivalent concentration of 2,6-dichlorophenolindophenol (DCPIP). As reported for Cu(II)2 (3,5-DIPS)4, addition of Mn3(3,5-DIPS)6 to this reaction mixture decreased the reduction of DCPIP without significantly affecting the oxidation of NADPH. The concentration of Mn3(3,5-DIPS)6 that produced a 50% decrease in DCPIP reduction (IC50) was found to be 5μM. Mechanistically, this inhibition of DCPIP reduction with ongoing NADPH oxidation by PHD was found to be due to the ability of Mn3(3,5-DIPS)6 to serve as a catalytic electron acceptor for reduced PHD as had been reported for Cu(II)2(3,5-DIPS)4. This catalytic decrease in reduction of DCPIP by Mn3(3,5-DIPS)6 was enhanced by the presence of a large concentration of DCPIP and decreased by the presence of a large concentration of NADPH, consistent with what had been observed for the activity of Cu(II)2(3,5-DIPS)4 Oxidation of NADPH by PHD in the presence of Mn3(3,5-DIPS)6 and the absence of DCPIP was linearly related to the concentration of added Mn3(3,5-DIPS)6 through the concentration range of 2.4 μM to 38μM with a 50% recovery of NADPH oxidation by PHD at a concentration of 6 μM Mn3(3,5-DIPS)6 Conversion of [3H] L-Arginine to [3H] L-Citrulline by purified rat brain nitric oxide synthase (NOS) was decreased in a concentrated related fashion with the addition of Mn3(3,5-DIPS)6 as well as Cu(II)2(3,5-DIPS)4 which is an extention of results reported earlier for Cu(II)2(3,5-DIPS)4. The concentration of these two compounds required to produce a 50% decrease in L-Citrulline synthesis by NOS, which may be due to down-regulation of NOS, were 0.1 mM and respectively, consistent with the relative potencies of these two complexes in preventing the reduction of Cytochrome c by NOS. It is concluded that Mn3(3,5-DIPS)6, as has been reported for Cu(II)2 (3,5-DIPS)4 , serves as an electron acceptor in down-regulating PHD and both of these complexes down-regulate rat brain NOS reactivity. A decrease in NO synthesis in animal models of seizure and radiation injury may account for the anticonvulsant, radioprotectant, and radiorecovery activities of Mn3(3,5-DIPS)6 and Cu(II)2(3,5-DIPS)4.