Use of Chimeric Forms of Neuronal Nitric‐Oxide Synthase as Dominant Negative Mutants

Abstract

SummaryBecause the functional form of neuronal nitric-oxide synthase (nNOS) is a homodimer, we investigated whether we could disrupt dimer formation with inactive nNOS chimeras acting as dominant negative mutants. To test this hypothesis, we either expressed the heme and reductase regions of rat nNOS as single domains or produced fusion proteins between the rat nNOS heme domain and various other electron-shuttling proteins. A dominant negative potential of these constructs was demonstrated by their ability to reduce NOS activity when transfected into a cell line stably expressing rat nNOS. In the presence of these nNOS mutant proteins, cellular levels of inactive nNOS monomers were signi cantly increased, indicating that their mechanism of action is through the disruption of nNOS dimer formation. These dominant negative mutants should prove valuable in analyzing the role of nNOS in biological systems. IUBMB Life, 48: 333-338, 1999

Neuronal nitric oxide synthase within paraventricular nucleus: blood pressure and baroreflex in two‐kidney, one‐clip hypertensive rats

“…Since the results from p725-and p739-injected rats were identical in all respects, the data were pooled and reported as the dominant negative nNOS group (DN nNOS). The sequences of these constructs and their synthesis have been published by our laboratories (Phung & Black, 1999). The rats were anaesthetized as before with ketamine and xylazine and secured in a stereotaxic apparatus with the skull levelled between bregma and lambda.…”

“…Low temperature (4 • C)-polyacrylamide gel electrophoresis was used to distinguish dimeric and monomeric forms of nNOS by methods previously used in our laboratory (Phung & Black, 1999). Samples containing 40 μg of protein each were loaded on an 8% polyacrylamide gel immediately after the addition of Laemelli sample buffer.…”

“…Results of densitometric analysis of three separate experiments are shown. For a description of constructs and their sequences see Phung & Black (1999). Values are means ± S.E.M.…”

“…Chronic inhibition of the generation of nitric oxide selectively within the PVN by a pharmacological approach is far from ideal, because it would block neuronal as well as endothelial sources. The dominant negative (DN) construct for neuronal nitric oxide synthase (nNOS) can very selectively decrease nitric oxide production in vitro by more than 90% (Phung & Black, 1999). These constructs have been expressed chronically in PVN after microinjection and functionally block nitric oxide suppression of the pressor response to injection of endothelin into the subfornical organ by 87% such that subsequent acute non-selective pharmacological inhibition by L-NAME did not elicit any additional significant increase in pressure (Rossi et al 2004).…”

Inhibition of Protein-tyrosine Phosphatases by Mild Oxidative Stresses Is Dependent on S-Nitrosylation

“…Dr. J. Pessin (42) provided wild type SHP-2. A NOS-1 dominant negative mutant (HemeRedF) has been described (11,43). Expression of HemeRedF in CHO cells inhibits radiation-induced endogenous NOS-1 activity (11).…”