Naphthoquinones and bioactive compounds from tobacco as modulators of neuronal nitric oxide synthase activity.

Studies were conducted with extracts of several varieties of tobacco in search of neuronal nitric oxide synthase (nNOS) inhibitors which may be of value in the treatment of stroke. Current therapies do not directly exploit modulation of nNOS activity due to poor selectivity of the currently available nNOS inhibitors. The properties of a potentially novel nNOS inhibitor(s) derived from tobacco extracts, and the concentration-dependent, modulatory effects of the tobacco-derived naphthoquinone compound, 2,3,6-trimethyl-1,4-naphthoquinone (TMN), on nNOS activity were investigated, using 2-methyl-1,4-naphthoquinone (menadione) as a control. Up to 31 microM, both TMN and menadione stimulated nNOS-catalysed L-citrulline production. However, at higher concentrations of TMN (62.5-500 microM), the stimulation was lost in a concentration-dependent manner. With TMN, the loss of stimulation did not decrease beyond the control activity. With menadione (62.5-500 microM), the loss of stimulation surpassed that of the control (78+/-0.01% of control activity), indicating a true inhibition of nNOS activity. This study suggests that potential nNOS inhibitors are present in tobacco, most of which remain to be identified.

Absence of nitric-oxide synthase in sequentially purified rat liver mitochondria.

Data, both for and against the presence of a mitochondrial nitric-oxide synthase (NOS) isoform, is in the refereed literature. However, irrefutable evidence has not been forthcoming. In light of this controversy, we designed studies to investigate the existence of the putative mitochondrial NOS. Using repeated differential centrifugation followed by Percoll gradient fractionation, ultrapure, never frozen rat liver mitochondria and submitochondrial particles were obtained. Following trypsin digestion and desalting, the mitochondrial samples were analyzed by nano-HPLC-coupled linear ion trap-mass spectrometry. Linear ion trap-mass spectrometry analyses of rat liver mitochondria as well as submitochondrial particles were negative for any peptide from any NOS isoform. However, recombinant neuronal NOS-derived peptides from spiked mitochondrial samples were easily detected, down to 50 fmol on column. The protein calmodulin (CaM), absolutely required for NOS activity, was absent, whereas peptides from CaM-spiked samples were detected. Also, l-[(14)C]arginine to l-[(14)C]citrulline conversion assays were negative for NOS activity. Finally, Western blot analyses of rat liver mitochondria, using NOS (neuronal or endothelial) and CaM antibodies, were negative for any NOS isoform or CaM. In conclusion, and in light of our present limits of detection, data from carefully conducted, properly controlled experiments for NOS detection, utilizing three independent yet complementary methodologies, independently as well as collectively, refute the claim that a NOS isoform exists within rat liver mitochondria.

NOx and R-NOx: effects on drug metabolism.

A family of three nitric oxide synthase 1(NOS) isoforms produces nitric oxide (NO*) and L-citrulline via a stepwise oxidation of the guanidinium nitrogen of L-arginine. Akin to cytochrome P450 (P450)-catalyzed monoxygenations, the NOS reactions are dependent on both molecular oxygen and NADPH. Although all three NOS isoforms (two constitutively-expressed isoforms; neuronal (nNOS) and endothelial (eNOS), and one inducible isoform; (iNOS)) produce identical products, the function of the NO* varies widely in terms of physiological functions due to the varied localization of the isoforms within different cell populations of the body. The NOS isoforms are homo-dimeric, bi-domain enzymes. Each monomer consisting of a flavin-containing reductase domain linked to a heme-containing oxygenase domain by a calmodulin (CaM)-binding sequence located between the two domains. The reductase domains of the NOS isoforms not only possess close sequence and structural homology to NADPH-cytochrome P450 oxidoreductase (CPOR), but also, owing to the flavins, resemble CPOR in function in that the reductase permits the passage of electrons from a NADPH-derived hydride, one at a time, to the NOS oxygenase domain. Although possessing very little structural resemblance to P450, the oxygenase domain of NOS is referred to as being "P450-like" due to the presence of iron protoporphyrin IX (heme), linked axially by a cysteine to the NOS protein, which carries out "P450-like" mono-oxygenation reactions. Considering the importance of NO* in regulating many physiological functions, the NOS isoforms are important drug targets. Modulators (both inhibitors as well as possible activators) of enzymatic function are needed to either decrease or increase NO* production, respectively, in various disease states. In addition to the widely known beneficial processes where NO* plays a role (i.e. neurotransmission, blood pressure regulation, immunomodulation), under certain conditions production of NO* (as well as other NOS-derived oxidants) can be detrimental by altering enzyme activity; for example P450-mediated drug metabolism. Furthermore, because of the close structural and functional similarities between NOS reductase and CPOR, many of the same redox-cycling and reductive reactions that occur with CPOR can also occur with NOS. The final focus of this review article is the less-well-recognized reactions mediated by NOS, which have recently begun to receive the attention they deserve. We hope to highlight reasons for concern regarding both altered drug metabolism as well as extra-hepatic, and target-organ toxicities in response to both altered NO* production as well as the detrimental interaction of NOS with certain xenobiotics.

Dinitrobenzene-mediated production of peroxynitrite by neuronal nitric oxide synthase.

Neuronal nitric oxide synthase (nNOS) is a modular enzyme that consists of a flavin-containing reductase domain and a heme-containing oxygenase domain, fused by a calmodulin (CaM)-binding sequence. Within the central nervous system, nNOS is localized in the cerebellum. CaM binding to nNOS activates both intradomain as well as interdomain electron transfer, and thus activity. The nNOS reductase shares many characteristics with NADPH-cytochrome P450 reductase (CPR), such as catalyzing the reduction of exogenous electron acceptors such as quinones and nitroarenes. The nitroarene 1,3-dinitrobenzene (1,3-DNB) is a cerebellar neurotoxicant in rats. 1,3-DNB is metabolized by CPR in liver, and it was proposed that metabolism of 1,3-DNB to reactive intermediates is involved in mediating the cerebellar neurotoxicity. We have found that, in a manner similar to CPR, nNOS can interact with 1,3-DNB and generate superoxide anion radical (O2*-). Electron transfer through the nNOS reductase is not limiting for nitric oxide (NO.) and L-citrulline production, even in the presence of certain exogenous electron acceptors such as 1,3-DNB. Therefore, NO., L-citrulline, and O2*- are simultaneously produced by nNOS in the presence of 1,3-DNB and other nitroarenes. The simultaneous production of NO. and O2*- leads to peroxynitrite (ONOO-) formation via the combination of these two radicals at a near-diffusion-controlled reaction rate. We present convincing data supporting the hypothesis that in the presence of 1,3-DNB, nNOS is converted from a purely NO. and L-citrulline synthase to a ONOO- and L-citrulline synthase, and propose that the resulting nitosative stress plays a role in the cerebellar neurotoxicity of 1,3-DNB. This paper introduces a new and novel enzymatic mechanism with direct toxicological implications whereby nNOS is converted into a ONOO- synthase by certain nitroarenes.