Differences in the activation of endothelial and neuronal nitric oxide synthase by oxidized calmodulin, and multiphoton activation of a photo-sensitive nitric oxide synthase inhibitor

The mammalian enzyme nitric oxide synthase (NOS) is responsible for the production of nitric oxide. Nitric oxide, a gaseous free radical, acts as a second messenger when trace amounts are produced by endothelial NOS (eNOS) and neuronal NOS (nNOS), and as a cytotoxin when high concentrations are produced by inducible NOS (iNOS). The regulation of eNOS and nNOS by the calcium-dependent binding of calmodulin (CAM) and the spatial and temporal inhibition of iNOS by a photolabile pro-drug were investigated.; Several CaM mutant proteins were engineered in an effort to identify the functional implications of individual methionines in CaM on the activity of nNOS and eNOS. The consequences of the site-specific oxidation of Met 144 and Met145 in CaM on the regulation of electron transfer within eNOS and nNOS was investigated. The results indicate that these amino acid side chains are involved in stabilizing the productive association between CaM and NOS. Furthermore, nNOS and cNOS are differentially regulated by the oxidation of specific methionine residues in CaM.; The cytotoxic level of nitric oxide produced from iNOS during some pathological conditions, such as arthritis, cerebral ischemia, and diabetes, exemplifies the need for a potent isoform-selective inhibitor of iNOS that can be spatially and temporally controlled. The slow, tight-binding iNOS selective inhibitor, 1400W, was found to inhibit iNOS activity by 50% at a concentration of 70 +/- 11 nM (IC50 value). Conjugating a coumarin-based, two-photon photolabile protecting group to 1400W produced a caged inhibitor or pro-drug. This caged inhibitor was a poor, slow-binding inhibitor of iNOS, giving an IC50 value of 1098 +/- 172 nM. Multiphoton laser scanning microscopy, used to photoactivate the caged inhibitor, confines the release of the drug to the small focal volume of the laser beam, approximately 1 mum3. The unmasked pro-drug released by multiphoton irradiation showed the same inhibitory properties and isoform specificity as 1400W (IC 50 value of 73 +/- 11 nM for iNOS).; Mammalian cell culture studies revealed that the caged inhibitor was not toxic to murine macrophage RAW 264.7 cells at therapeutic concentrations. Multiphoton irradiation of the caged compound on the microscope stage inhibited iNOS derived nitrite production from bacterial lipopolysaccharide stimulated RAW 264.7 cells. In contrast, the caged inhibitor showed no inhibition of nitrite production from lipopolysaccharide induced RAW 264.7 cells. The demonstration of the use of multiphoton irradiation to photorelease a therapeutic concentration of 1400W from the caged inhibitor without affecting the viability of the RAW 264.7 cells shows the potential of this technology. The use of multiphoton laser scanning microscopy and a caged iNOS selective inhibitor may allow effective spatial and temporal control of iNOS activity, thereby decreasing the harmful consequences of nitric oxide production associated with several pathologies.