B.S.S. Masters, P. Martasek, L.J. Roman, R.T. Miller, C.S. Raman*, T.J. Poulos*,H. Li*, and J.S. Nishimura
University of Texas Health Science Center at San Antonio, TX and *University of California, Irvine, CA
The isoforms of nitric oxide synthase are known as neuronal (nNOS), inducible (iNOS), and endothelial (eNOS) and are encoded by distinct genes. Not only do they differ in molecular weight, with the nNOS being the largest, but the functions of their common product, NO. also differ. Since all three isoforms utilize L-arginine (L-arg) to produce L-citrulline (L-cit) and NO., understanding those properties which determine the localization and function of these enzymes is important. Utilizing a protease-negative (lon-) BL21 E. coli cell line, we have expressed large quantities of all three isoforms in order to study their structure-function relationships. The production of modules containing only heme, flavin, or binding site(s) for tetrahydrobiopterin (BH4) and/or other cofactors has permitted the determination of spectroscopic and binding parameters and the effects of cofactors on O2 reduction, as well as crystallization of several constructs. Although work is in progress on the latter, characterization of the cysteine331alanine (C331A) mutant of nNOS has revealed a protein with altered binding characteristics for L-arg and BH4. This mutant and a flavoprotein construct derived from nNOS have been utilized to show that the flavoprotein domain is necessary and sufficient for superoxide anion production from nNOS [Miller et al. (1997) Biochemistry, 36, 15277-15284]. Despite the inactivity of this mutant in its isolated form, overnight incubation with high concentrations of L-arg and BH4 converts it to a fully active enzyme with a turnover number equaling that of the wild-type enzyme. Recent experiments, examining the kinetics of binding and release of CO from the heme of various NOS constructs using flash photolysis, verify that the off-rate for CO binding to the C331A mutant is very high, producing an unstable reduced, CO difference spectrum. A calmodulin-minus mutant of eNOS has been expressed and both intact and heme domain constructs have been purified in large quantities. These and other mutant and modules are being examined by crystallographic methods.
Supported by NIH Grant No. GM 52419 & Robert A. Welch Foundation Grant No. AQ-1192 to B.S.S.M.
Steven Gross
Cornell University Medical College, New York, NY 10021, USA
Vascular dysfunctions and acute mortality associated with septic shock have been attributed to NO overproduction in the blood vessel wall. Therefore, therapeutic strategies which limit NO may be lifesaving in septic patients. My presentation will focus on the pathway for biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor of NO synthase (NOS), as a potential therapeutic target for limiting the toxicity of NO excess. Notably, cytokines and bacterial-derived immuno-stimulants that trigger iNOS gene expression in vascular smooth muscle cells (SMC) also elicit expression of the first and rate-limiting enzyme of BH4 synthesis, GTP cyclohydrolase I (GTPCH). Earlier, we demonstrated that NO synthesis in SMC is abolished by inhibitors of GTPCH and restored by administering excess BH4; this demonstrates that BH4 availability is rate-limiting to NO synthesis in vitro. We have now found that stable overexpression of GTPCH cDNA in SMC results in elevated levels of intracellular BH4 and a 2-3 fold increase in immunostimulant-evoked NO synthesis, on a per cell basis. This finding supports the view that GTPCH is rate-limiting to BH4 synthesis and hence NO production in vitro. Analysis of reporter constructs containing DNA sequences 5' upstream of the rat GTPCH gene, reveal that upregulation of GTPCH gene expression by immunostimulants occurs at the level of transcription. It is significant, however, that additional post-translational mechanisms for modulation of GTPCH activity appear also to operate. We further show that GTPCH is upregulated by immunostimulants in the rat in vivo, where BH4 levels limit NO synthesis, vascular dysfunctions and killing. Together, our findings indicate that BH4 availability limits NO overproduction in vitro and in vivo and suggest that BH4 serves as a transcellular metabolite that can be produced by one cell type for high output NO synthesis by another.
Support: HL50656 and 40403
ATP- and GTP-dependent activation of iron regulatory protein-1 by oxidative stress
Kostas Pantopoulos and Matthias W. Hentze
European Molecular Biology Laboratory, Meyerhofstrasse 1 D-69117 Heidelberg, Germany
Iron regulatory protein-1 (IRP-1), the cytoplasmic post-transcriptional regulator of cellular iron metabolism, is rapidly activated by oxidative stress to bind to mRNA iron-responsive elements (IREs). We have reconstituted the response of IRP-1 to extracellular H2O2 in a cell-free system derived from murine B6 fibroblasts. Our protocol allows to separate the cytosol from the remainder of the cells (cell pellet). IRP-1 in the cytosolic fraction fails to be directly activated by addition of H2O2. IRP-1 activation requires the presence of a non-soluble, possibly membrane-associated component in the cell pellet. The cell-free system faithfully recapitulates characteristic hallmarks of IRP-1 activation by H2O2 in intact cells. We show that the H2O2-mediated activation of IRP-1 is temperature-dependent and blocked by addition of ATP-gS and GTP-gS (but not by ATP and GTP). IRP-1 activation is also sensitive to treatment with calf intestinal alkaline phosphatase, and to depletion of cytosolic ATP with hexokinase and glucose. The in vitro reconstitution of this oxidative stress-induced pathway has opened a new avenue for the biochemical dissection of the regulation of mammalian iron metabolism by oxidative stress. Our data show that H2O2 serves as signal that must be sensed to stimulate an ATP and GTP-dependent pathway to activate IRP-1.
Regulation of mitochondrial oxygen uptake by O2 and NO
Alberto Boveris, Lidia E. Costa, and Juan J. Poderoso
Laboratory of Free Radical Biology, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina
Mitochondria in physiological conditions operate at about 3 to 20 µM O2. The dependence of the respiration rate of mitochondria and isolated cellson [O2] is described by an hyperbola; therefore it is conveniently described by [O2]0.5, the oxygen concentration that provides half-maximal respiration rate. The [O2]0.5, determined by high-resolution respirometry, for liver and heart mitochondria in state 4 (succinate rotenone and malate-pyruvate as substrates, respectively) were 0.30 ± 0.06 and 0.25 ± 0.05 µM O2, respectively. In active state 3 (+ ADP) the [O2]0.5 were 1.7 ± 0.1 and 1.5 ± 0.1 µM O2 for liver and heart mitochondria. Taking strict Michaelis-Menten kinetics it follows that limitation of active mitochondrial respiration may occur at physiological tissue pO2 (a critical rate of 80 % maximal respiratory rate will be reached at 6 µM O2 Nitric oxide inhibits cytochrome oxidase activity and respiration in isolated mitochondria, with half-maximal effect at 0.1 - 0.3 µM NO; NO binding to cytocrome oxidase is reversible and competitive with O2 (160 times higher affinity for NO than for O2). Then, an estimated physiological [NO] of 50 nM will be able to inhibit cytochrome oxidase activity by about 50 % in the presence of 8 µM O2. Nitric oxide also inhibits cytochrome b to cytochrome c electron transfer (half-maximal effect at 0.35 µM NO) and increases mitochondrial O2. production. The NO-induced generation of O2. may remove NO inhibition of cytochrome oxidase and set a feed-back mechanism to regulate mitochondrial oxygen uptake. It is then apparent than a complex relationship exists between NO and O2 steady state concentrations that is able to regulate mitochondrial oxygen uptake. Decreased tissue pO2 activates endothelial NOS whereas NO produced by the endothelium or mitochondria (C. Giulivi and C. Richter, personal communications) will be able to regulate O2 uptake.
Reactive metabolites of oxygen and nitrogen, NF-kB, and chronic inflammation
Matthew B. Grisham
Department of Molecular and Cellular Physiology, LSU Medical Center Shreveport, LA
Reactive metabolites of oxygen and nitrogen have been suggested to directly or indirectly promote inflammatory tissue injury via their ability to oxidize and degrade essential cellular constituents. More recent work suggests that reactive species may be equally important as signaling agents with the ability to modulate gene expression via activation of certain transcription factors such as NF-kB. NF-kB is a ubiquitous transcription factor activated by certain oxidants, lipid mediators and/or pro inflammatory cytokines via the degradation of its complexes inhibitor IkB. Translocation of NF-kB into the nucleus upregulates the transcription of a variety of adhesion molecules, cytokines and enzymes. Because the proteolytic degradation of the post-translationally modified IkB is known to be mediated by the 26S proteasome complex, we wished to assess the therapeutic antiinflammatory activity of a selective protea-some inhibitor in a model of chronic polyarthritis. Chronic polyarthritis with granulomatous liver inflammation was induced in female Lewis rats via the intraperitoneal injection of peptidoglycan polysaccharide (PG/PS; 25 µg/g body weight). Twenty rats were randomized into 3 groups consisting of a saline-injected control group, a PG/PS arthritic group given vehicle (methylcellulose) p.o. daily beginning 7 days following the induction of arthritis, and a PG/PS arthritic group given 0.3 mg/kg/day MG/341 (proteasome inhibitor; Ki = 1.2 nM) p.o. daily beginning 7 days following the induction of arthritis. Arthritic symptoms were monitored throughout the course of the study and were quantified using the total arthritis index score and measurement of paw volume. Twenty eight days following the induction of arthritis, animals were sacrificed and inflamed organs (liver and paws) were retrieved for histological and biochemical analysis. In a first series of experiments, we found that MG-341 inhibited, in a dose-dependent manner the TNF-induced activation of NF-kB in human endothelial cells in vitro. In addition, we found that certain antioxidants or MG-341 significantly inhibited the TNF-induced surface expression of ICAM-1, E-selectin, and VCAM-1 in HUVECs as well as inhibited the PG/PS induced upregulation of iNOS in a murine macrophage cell line in vitro. We also found that oral administration of MG-341 attenuated polyarthritis induced by the intraperitoneal injection of PG/PS as assessed by significant induction in the total arthritis and average hind paw volume. Histologically, drug treatment attenuated the cellular infiltration, synovial thickening and pannus formation as well as the bone and cartilage erosion typical of PG/PS induced arthritis. The inhibition of polyarthritis correlated with significant reductions in plasma levels of nitrate and nitrite and IL-6 compared to vehicle treated controls. The gross liver inflammation score was also attenuated by drug treatment. Furthermore, PG/PS-induced upregulation of liver inducible nitric oxide synthase and VCAM-1 was significantly attenuated by therapeutic treatment with MG-341. We conclude that the 26S proteasome and thus NF kB play important roles in regulating the acute and chronic inflammatory responses in vivo.