MOLECULAR ACTIONS OF A MN(III)PORPHYRIN SUPEROXIDE-DISMUTASE MIMETIC AND PEROXYNITRITE SCAVENGER - REACTION WITH NITRIC-OXIDE AND DIRECT INHIBITION OF NO SYNTHASE AND SOLUBLE GUANYLYL CYCLASE

Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), described as a superoxide dismutase mimetic and peroxynitrite scavenger, has been us ed previously to investigate the cytotoxic potential of superoxide and peroxynitrite in several pathological models. Here we report on the i nterference of MnTMPyP with NO/cGMP signaling using cultured endotheli al cells as well as purified soluble guanylyl cyclase (sGC) either act ivated by the NO donor 2,2-diethyl-1-nitroso-oxyhydrazine sodium salt (DEA/NO) or reconstituted with nitric oxide synthase (NOS). MnTMPyP in hibited endothelial cGMP accumulation induced by A23187 (0.3 mu M) wit h an IC50 of 75.0 +/- 10.4 mu M but had no significant effect on the p otency of the Ca2+ ionophore. Purified NOS was inhibited by MnTMPyP (I C50 = 5.5 +/- 0.8 mu M) because of an interference of the Mn-porphyrin with the reductase domain of the enzyme. The most pronounced actions of MnTMPyP were direct inhibition of sGC and scavenging of NO. Purifie d sGC stimulated with either Ca2+/calmodulin-activated NOS (in the pre sence of GSH) or DEA/NO (in the absence of GSH) was inhibited with IC5 0 values of 0.8 +/- 0.09 mu M and 0.6 +/- 0.2 mu M, respectively. In t he presence of GSH, MnTMPyP was reduced to the Mn(II) complex, resulti ng in efficient scavenging of NO under these conditions. Our data demo nstrate that MnTMPyP (i) interferes with the reductase domain of NOS, (ii) scavenges NO in the presence of GSH, and (iii) is a potent direct inhibitor of sGC. These results cast doubt on the usefulness of MnTMP yP and related Mn-porphyrin complexes as probes to study the involveme nt of peroxynitrite/superoxide in biological systems.