Cobalamin in inflammation III — glutathionylcobalamin and methylcobalamin/adenosylcobalamin coenzymes: the sword in the stone? How cobalamin may directly regulate the nitric oxide synthases

Several mysteries surround the structure and function of the nitric oxide synthases (NOS). The NOS oxygenase domain structure is unusually open with a large area of solvent that could accommodate an unidentified ligand. The exact mechanism of the two-step five-electron monoxygenation of arginine to NG-hydroxy-L-arginine, thence to citrulline and nitric oxide (NO), is not clear, particularly as arginine/NG-hydroxy-L-arginine is bound at a great distance to the supposed catalytic heme Fe [III], as the anti-stereoisomer. The Return of the Scarlet Pimpernel Paper proposed that cobalamin is a primary indirect regulator of the NOS. An additional direct regulatory effect of the ‘base-off’ dimethylbenzimidazole of glutathionylcobalamin (GSCbl), which may act as a sixth ligand to the heme iron, promote Co-oriented, BH4/BH3 radical catalysed oxidation of L-arginine to NO, and possibly regulate the rate of inducible NOS/NO production by the NOS dimers, is further advanced. The absence of homology between the NOS and methionine synthase/methylmalonyl CoA mutase may enable GSCbl to regulate both sets of enzymes simultaneously by completely separate mechanisms. Thus, cobalamin may exert central control over both pro-and anti-inflammatory systems

Macrophage migration inhibitory factor-nitric oxide interaction in human fetal membranes at term pregnancy

OBJECTIVES: Macrophage migration inhibitory factor (MIF), a multifunctional proinflammatory cytokine, has been recently involved in many aspects of reproduction including pregnancy. However, no evidence is available on the role of MIF in gestational tissues nor on factors regulating MIF production. This study, conducted on explants of human fetal membranes at term gestation, has been undertaken to investigate whether: (1) MIF is produced by fetal membranes; (2) nitric oxide (NO) can regulate local MIF production; and (3) MIF, in turn, can influence NO release in these tissues. METHODS: Tissues were obtained from 56 healthy women who underwent elective cesarean delivery. Fetal membranes have been incubated with either sodium nitroprusside (NP), a NO donor, or recombinant MIF (r-MIF), or a specific anti-MIF antibody (MIF-Ab). Reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot, enzyme-linked immunosorbent assay (ELISA), and colorimetric assay have been used to detect MIF mRNA and protein, inducible nitric oxide syntase (iNOS), and NO metabolites. RESULTS: Fetal membranes basally express MIF mRNA and protein and release MIF. Exposing tissues to NP results in an increase of MIF mRNA expression and protein release. Conversely, treatment of tissues with MIF is followed by a reduction in iNOS mRNA and protein expression as well as in NO release. These effects are reversed by adding MIF-Ab. CONCLUSIONS: MIF is generated and released by human fetal membranes at term. MIF mRNA and protein expression and release are modulated by NO. MIF, in turn, can reduce iNOS expression and NO release by these tissues. NO could be a regulator of MIF production in pregnancy and labor