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 5 μM
. 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)4Oxidation 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 38 μM
with a 50% recovery of NADPH oxidation by PHD at a concentration of 6 μM Mn3(3,5-DIPS)6Conversion 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 8μM
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
.
