Inverse relationship between markers of nitric oxide formation and plasma matrix metalloproteinase-9 levels in healthy volunteers

Background: Nitric oxide (NO) is a major regulator of cardiovascular homeostasis and has anti-atherogenic properties. Reduced NO formation is associated with endothelial dysfunction and with cardiovascular risk factors. Although NO downregulates the expression and activity of the pro-atherogenic enzyme matrix metalloproteinase-9 (MMP-9), no previous clinical study has examined whether endogenous NO formation is inversely associated with the circulating levels of pro-MMP-9, which are associated with cardiovascular events. We examined this hypothesis in 175 healthy male subjects who were non-smokers. Methods: To assess NO bioavailability, the plasma concentrations of nitrite, nitrate, and cGMP were determined using an ozone-based chemiluminescence assay and an enzyme immunoassay. Pro-MMP-9 and pro-MMP-2 levels were measured in plasma samples by gelatin zymography. Results: We found significant negative correlations between pro-MMP-9 levels and plasma nitrite (P=0.035, rs=-0.159), nitrate (P=0.040, rs=-0.158), and cGMP (P=0.011, rs=-0.189) concentrations. However, no significant correlations were found between pro-MMP-2 levels and the plasma concentrations of markers of NO bioavailability (all P>0.05). Conclusions: There is an inverse relationship between markers of NO formation and plasma MMP-9 levels. This finding may shed some light on the possible mechanisms involved in the increased cardiovascular risk of apparently healthy subjects with low NO bioavailability or high circulating levels of pro-MMP-9. (C) 2008 Elsevier B.V. All rights reserved.

Vascular smooth muscle relaxation mediated by nitric oxide donors: a comparison with acetylcholine, nitric oxide and nitroxyl ion

I Vasorelaxant properties of three nitric oxide (NO) donor drugs (glyceryl trinitrate, sodium nitroprusside and spermine NONOate) in mouse aorta (phenylephrine pre-contracted) were compared with those of endothelium-derived NO (generated with acetylcholine), NO free radical (NO; NO gas solution) and nitroxyl ion (NO-; from Angeli's salt). 2 The soluble guanylate cyclase inhibitor, ODQ (1H-(1,2,4-)oxadiazolo(4,3-a)-quinoxalin-1-one; 0.3, 1 and 10 muM), concentration-dependently inhibited responses to all agents. 10 muM ODQ abolished responses to acetylcholine and glyceryl trinitrate, almost abolished responses to sodium nitroprusside but produced parallel shifts (to a higher concentration range; no depression in maxima) in the concentration-response curves for NO gas solution, Angeli's salt and spermine NONOate. 3 The NO scavengers, carboxy-PTIO, (2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-indazoline-1-oxyl-3-oxide; 100 muM) and hydroxocobalamin (100 muM), both inhibited responses to NO gas solution and to the three NO donor drugs, but not Angeli's salt. Hydroxocobalamin, but not carboxy-PTIO, also inhibited responses to acetylcholine. 4 The NO- inhibitor, L-cysteine (3 mm), inhibited responses to Angeli's salt, acetylcholine and the three NO donor drugs, but not NO gas solution. 5 The data suggest that, in mouse aorta, responses to all three NO donors involve (i) activation of soluble guanylate cyclase, but to differing degrees and (ii) generation of both NO and NO-. Glyceryl trinitrate and sodium nitroprusside, which generate NO following tissue bioactivation, have profiles resembling the profile of endothelium-derived NO more than that of exogenous NO. Spermine NONOate, which generates NO spontaneously outside the tissue, was the drug that most closely resembled (but was not identical to) exogenous NO.

Expression of asparagine synthetase in response to carbohydrate supply in model callus cultures and shoot tips of asparagus (Asparagus officinalis L.)

Sugar uptake and metabolism were studied in callus cultures and shoot tips of asparagus. Asparagus callus cultures were used to model senescence in shoot tips. Callus cultures absorbed glucose from a nutrient medium, and accumulated sucrose, glucose and fructose. This uptake of glucose by the callus cultures down-regulated expression of asparagine synthetase and beta -galactosidase transcripts that otherwise accumulated when sugar was withheld. When 80 mm-long asparagus shoots were excised from growing plants and placed in 2% and 8% sucrose solutions, endogenous concentrations of sucrose, glucose, fructose, UDPglucose, and glucose-6-phosphate declined in the 30mm-long meristematic tip regions. At the same time, asparagine and asparagine synthetase gene transcripts began to accumulate in these tips. When 10 mm-long asparagus shoot tips were placed on glucose- or fructose-containing agar, the tips accumulated sucrose, glucose and fructose, and asparagine accumulation and expression of asparagine synthetase were marginally reduced. We concluded that in callus cultures, asparagine synthetase expression was sugar regulated, but that sugar regulation was not as pronounced in asparagus shoot tips. This may be due in part to slower rates of sugar uptake into shoot tips and in part to compartmentation of sugars in the tips. We suggest that callus cultures are not a suitable model for metabolic studies in asparagus shoot tips.

GLUT4 - At the cross roads between membrane trafficking and signal transduction

GLUT4 is a mammalian facilitative glucose transporter that is highly expressed in adipose tissue and striated muscle. In response to insulin, GLUT4 moves from intracellular storage areas to the plasma membrane, thus increasing cellular glucose uptake. While the verification of this 'translocation hypothesis' (Cushman SW. Wardzala LJ. J Biol Chem 1980;255: 4758-4762 and Suzuki K, Kono T. Proc Natl Acad Sci 1980;77: 2542-2545) has increased our understanding of insulin-regulated glucose transport, a number of fundamental questions remain unanswered. Where is GLUT4 stored within the basal cell? How does GLUT4 move to the cell surface and what mechanism does insulin employ to accelerate this process) Ultimately we require a convergence of trafficking studies with research in signal transduction. However, despite more than 30 years of intensive research we have still not reached this point. The problem is complex, involving at least two separate signal transduction pathways which feed into what appears to be a very dynamic sorting process. Below we discuss some of these complexities and highlight new data that are bringing us closer to the resolution of these questions.

Crystal structure of yeast acetohydroxyacid synthase: A target for herbicidal inhibitors

Acetohydroxyacid synthase (AHAS; EC 4.1.3.18) catalyzes the first step in branched-chain amino acid biosynthesis. The enzyme requires thiamin diphosphate and FAD for activity, but the latter is unexpected, because the reaction involves no oxidation or reduction. Due to its presence in plants, AHAS is a target for sulfonylurea and imidazolinone herbicides. Here, the crystal structure to 2.6 A resolution of the catalytic subunit of yeast AHAS is reported. The active site is located at the dimer interface and is near the proposed herbicide-binding site. The conformation of FAD and its position in the active site are defined. The structure of AHAS provides a starting point for the rational design of new herbicides. (C) 2002 Elsevier Science Ltd.

Skeletal muscle mitochondrial ATP production rate and walking performance in peripheral arterial disease

This study tested the hypotheses that skeletal muscle mitochondrial ATP production rate (MAPR) is impaired in patients with peripheral arterial disease (PAD) and that it relates positively to their walking performances. Seven untrained patients, eight exercise-trained patients and 11 healthy controls completed a maximal walking test and had muscle sampled from the gastrocnemius medialis muscle. Muscle was analysed for its MAPR in the presence of pyruvate, palmitoyl-L-carnitine or both, as well as citrate synthase (CS) activity. MAPRs were not different between untrained PAD and controls. In contrast, MAPRs (pyruvate) were significantly higher in trained PAD vs. controls. MAPR (pyruvate combinations) was also significantly higher in trained than untrained PAD muscle. MAPR and CS activity were highly correlated with walking performance in patients, but not in controls. These data do not support the hypothesis that isolated mitochondria are functionally impaired in PAD and demonstrate that the muscle mitochondrial capacity to oxidize carbohydrate is positively related to walking performance in these patients.

Crystallization of the FAD-independent acetolactate synthase of Klebsiella pneumoniae

Leucine and valine are formed in a common pathway from pyruvate in which the first intermediate is 2-acetolactate. In some bacteria, this compound also has a catabolic fate as the starting point for the butanediol fermentation. The enzyme (EC 4.1.3.18) that forms 2-acetolactate is known as either acetohydroxyacid synthase (AHAS) or acetolactate synthase (ALS), with the latter name preferred for the catabolic enzyme. A significant difference between AHAS and ALS is that the former requires FAD for catalytic activity, although the reason for this requirement is not well understood. Both enzymes require the cofactor thiamine diphosphate. Here, the crystallization and preliminary X-ray diffraction analysis of the Klebsiella pneumoniae ALS is reported. Data to 2.6 Angstrom resolution have been collected at 100 K using a rotating-anode generator and an R-AXIS IV++ detector. Crystals have unit-cell parameters a = 137.4, b = 143.9, c = 134.4 Angstrom, alpha = 90, beta = 108.4, gamma = 90degrees and belong to space group C2. Preliminary analysis indicates that there are four monomers located in each asymmetric unit.

Regulatory interactions in Arabidopsis thaliana acetohydroxyacid synthase

Acetohydroxyacid synthase (AHAS; EC 4.1.3.18) contains catalytic and regulatory subunits, the latter being required for sensitivity to feedback regulation by leucine, valine and isoleucine. The regulatory subunit of Arabidopsis thaliana AHAS possesses a sequence repeat and we have suggested preciously that one repeat binds leucine while the second binds valine or isoleucine, with synergy between the two sites. We have mutated four residues in each repeat, based on a model of the regulatory subunit. The data confirm that there are separate leucine and valine/isoleucine sites, and suggest a complex pathway for regulatory signal transmission to the catalytic subunit. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Vascular dysfunction and heart failure: Epiphenomenon or etiologic agent?

Endothelial function plays a key role in the local regulation of vascular tone. Alterations in endothelial function may result in impaired release of endothelium-derived relaxing factors or increased release of endothelium-derived contracting factors. Heart failure may impair endothelial function by means of reduced synthesis and release of nitric oxide (NO) or by increased degradation of NO and increased production of endothelin-1. Endothelial dysfunction may worsen heart function by means of peripheral effects, causing increased afterload and central effects such as myocardial ischemia and inducible nitric oxide synthase (iNOS)-induced detrimental effects. Evidence from clinical studies has suggested that there is a correlation between decreased endothelial function and increasing severity of congestive heart failure (CHF). Treatments that improve heart function may also improve endothelial dysfunction. The relationship between endothelial dysfunction and heart failure may be masked by the stage of endothelial dysfunction, the location of vessels being tested, and the state of endothelial-dependent vasodilatation response.

In vivo and in vitro models demonstrate a role for caveolin-1 in the pathogenesis of ischaemic acute renal failure

Caveolae and their proteins, the caveolins, transport macromolecules; compartmentalize signalling molecules; and are involved in various repair processes. There is little information regarding their role in the pathogenesis of significant renal syndromes such as acute renal failure (ARF). In this study, an in vivo rat model of 30 min bilateral renal ischaemia followed by reperfusion times from 4 h to 1 week was used to map the temporal and spatial association between caveolin-1 and tubular epithelial damage (desquamation, apoptosis, necrosis). An in vitro model of ischaemic ARF was also studied, where cultured renal tubular epithelial cells or arterial endothelial cells were subjected to injury initiators modelled on ischaemia-reperfusion (hypoxia, serum deprivation, free radical damage or hypoxia-hyperoxia). Expression of caveolin proteins was investigated using immunohistochemistry, immunoelectron microscopy, and immunoblots of whole cell, membrane or cytosol protein extracts. In vivo, healthy kidney had abundant caveolin-1 in vascular endothelial cells and also some expression in membrane surfaces of distal tubular epithelium. In the kidneys of ARF animals, punctate cytoplasmic localization of caveolin-1 was identified, with high intensity expression in injured proximal tubules that were losing basement membrane adhesion or were apoptotic, 24 h to 4 days after ischaemia-reperfusion. Western immunoblots indicated a marked increase in caveolin-1 expression in the cortex where some proximal tubular injury was located. In vitro, the main treatment-induced change in both cell types was translocation of caveolin-1 from the original plasma membrane site into membrane-associated sites in the cytoplasm. Overall, expression levels did not alter for whole cell extracts and the protein remained membrane-bound, as indicated by cell fractionation analyses. Caveolin-1 was also found to localize intensely within apoptotic cells. The results are indicative of a role for caveolin-1 in ARF-induced renal injury. Whether it functions for cell repair or death remains to be elucidated. Copyright (C) 2003 John Wiley Sons, Ltd.

Molecular basis of sulfonylurea herbicide inhibition of acetohydroxyacid synthase

Acetohydroxyacid synthase (AHAS) (acetolactate synthase, EC 4.1.3.18) catalyzes the first step in branchedchain amino acid biosynthesis and is the target for sulfonylurea and imidazolinone herbicides. These compounds are potent and selective inhibitors, but their binding site on AHAS has not been elucidated. Here we report the 2.8 Angstrom resolution crystal structure of yeast AHAS in complex with a sulfonylurea herbicide, chlorimuron ethyl. The inhibitor, which has a K-i of 3.3 nM blocks access to the active site and contacts multiple residues where mutation results in herbicide resistance. The structure provides a starting point for the rational design of further herbicidal compounds.

Systematic characterization of mutations in yeast acetohydroxyacid synthase: Interpretation of herbicide-resistance data

Acetohydroxyacid synthase (AHAS, EC 4.1.3.18) catalyses the first step in branched-chain amino acid biosynthesis and is the target for sulfonylurea and imidazolinone herbicides, which act as potent and specific inhibitors. Mutants of the enzyme have been identified that are resistant to particular herbicides. However, the selectivity of these mutants towards various sulfonylureas and imidazolinones has not been determined systematically. Now that the structure of the yeast enzyme is known, both in the absence and presence of a bound herbicide, a detailed understanding of the molecular interactions between the enzyme and its inhibitors becomes possible. Here we construct 10 active mutants of yeast AHAS, purify the enzymes and determine their sensitivity to six sulfonylureas and three imidazolinones. An additional three active mutants were constructed with a view to increasing imidazolinone sensitivity. These three variants were purified and tested for their sensitivity to the imidazolinones only. Substantial differences are observed in the sensitivity of the 13 mutants to the various inhibitors and these differences are interpreted in terms of the structure of the herbicide-binding site on the enzyme.