Chert oxygen isotope ratios are driven by Earth's thermal evolution.

The 18O/16O ratio of cherts (δ18Ochert) increases nearly monotonically by ~15‰ from the Archean to present. Two end-member explanations have emerged: cooling seawater temperature (TSW) and increasing seawater δ18O (δ18Osw). Yet despite decades of work, there is no consensus, leading some to view the δ18Ochert record as pervasively altered. Here, we demonstrate that cherts are a robust archive of diagenetic temperatures, despite metamorphism and exposure to meteoric fluids, and show that the timing and temperature of quartz precipitation and thus δ18Ochert are determined by the kinetics of silica diagenesis. A diagenetic model shows that δ18Ochert is influenced by heat flow through the sediment column. Heat flow has decreased over time as planetary heat is dissipated, and reasonable Archean-modern heat flow changes account for ~5‰ of the increase in δ18Ochert, obviating the need for extreme TSW or δ18Osw reconstructions. The seawater oxygen isotope budget is also influenced by solid Earth cooling, with a recent reconstruction placing Archean δ18OSW 5 to 10‰ lower than today. Together, this provides an internally consistent view of the δ18Ochert record as driven by solid Earth cooling over billion-year timescales that is compatible with Precambrian glaciations and biological constraints and satisfyingly accounts for the monotonic nature of the δ18Ochert trend.

Coupled partitioning of Au and As into pyrite controls formation of giant Au deposits.

The giant Carlin-type Au deposits (Nevada, USA) contain gold hosted in arsenic-rich iron sulfide (pyrite), but the processes controlling the sequestration of Au in these hydrothermal systems are poorly understood. Here, we present an experimental study investigating the distribution of Au and As between hydrothermal fluid and pyrite under conditions similar to those found in Carlin-type Au deposits. We find that Au from the fluid strongly partitions into a newly formed pyrite depending on the As concentration and that the coupled partitioning behavior of these two trace elements is key for Au precipitation. On the basis of our experimentally derived partition coefficients, we developed a mass balance model that shows that simple partitioning (and the underlying process of adsorption) is the major depositional process in these systems. Our findings help to explain why pyrite in Carlin-type gold deposits can scavenge Au from hydrothermal fluids so efficiently to form giant deposits.

Effect of soluble guanylyl cyclase activator and stimulator therapy on nitroglycerin-induced nitrate tolerance in rats.

Chronic nitroglycerin (GTN) anti-ischemic therapy induces side effects such as nitrate tolerance and endothelial dysfunction. Both phenomena could be based on a desensitization/oxidation of the soluble guanylyl cyclase (sGC). Therefore, the present study aims at investigating the effects of the therapy with the sGC activator BAY 60-2770 and the sGC stimulator BAY 41-8543 on side effects induced by chronic nitroglycerin treatment. Male Wistar rats were treated with nitroglycerin (100mg/kg/d for 3.5days, s.c. in ethanol) and BAY 60-2770 (0.5 or 2.5mg/kg/d) or BAY 41-8543 (1 and 5mg/kg/d) for 6days. Therapy with BAY 60-2770 but not with BAY 41-8543 improved nitroglycerin-triggered endothelial dysfunction and nitrate tolerance, corrected the decrease in aortic nitric oxide levels, improved the cGMP dependent activation of protein kinase I in aortic tissue and reduced vascular, cardiac and whole blood oxidative stress (fluorescence and chemiluminescence assays; 3-nitrotyrosine staining). In contrast to BAY 41-8543, the vasodilator potency of BAY 60-2770 was not impaired in isolated aortic ring segments from nitrate tolerant rats. sGC activator therapy improves partially the adverse effects of nitroglycerin therapy whereas sGC stimulation has only minor beneficial effects pointing to a nitroglycerin-dependent sGC oxidation/inactivation mechanism contributing to nitrate tolerance.

Articular cartilage degeneration classification by means of high-frequency ultrasound.

CONTEXT: To date only single ultrasound parameters were regarded in statistical analyses to characterize osteoarthritic changes in articular cartilage and the potential benefit of using parameter combinations for characterization remains unclear. OBJECTIVE: Therefore, the aim of this work was to utilize feature selection and classification of a Mankin subset score (i.e., cartilage surface and cell sub-scores) using ultrasound-based parameter pairs and investigate both classification accuracy and the sensitivity towards different degeneration stages. DESIGN: 40 punch biopsies of human cartilage were previously scanned ex vivo with a 40-MHz transducer. Ultrasound-based surface parameters, as well as backscatter and envelope statistics parameters were available. Logistic regression was performed with each unique US parameter pair as predictor and different degeneration stages as response variables. The best ultrasound-based parameter pair for each Mankin subset score value was assessed by highest classification accuracy and utilized in receiver operating characteristics (ROC) analysis. RESULTS: The classifications discriminating between early degenerations yielded area under the ROC curve (AUC) values of 0.94-0.99 (mean ± SD: 0.97 ± 0.03). In contrast, classifications among higher Mankin subset scores resulted in lower AUC values: 0.75-0.91 (mean ± SD: 0.84 ± 0.08). Variable sensitivities of the different ultrasound features were observed with respect to different degeneration stages. CONCLUSIONS: Our results strongly suggest that combinations of high-frequency ultrasound-based parameters exhibit potential to characterize different, particularly very early, degeneration stages of hyaline cartilage. Variable sensitivities towards different degeneration stages suggest that a concurrent estimation of multiple ultrasound-based parameters is diagnostically valuable. In-vivo application of the present findings is conceivable in both minimally invasive arthroscopic ultrasound and high-frequency transcutaneous ultrasound.

Ultrasonic attenuation estimation of the pregnant cervix: a preliminary report.

OBJECTIVE: Estimates of ultrasonic attenuation (the loss of energy as an ultrasonic wave propagates through tissue) have been used to evaluate the structure and function of tissues in health and disease. The purpose of this research was to develop a method to estimate ultrasonic cervical attenuation during human pregnancy using a clinical ultrasound system. METHODS: Forty women underwent a cervical scan once during pregnancy with the Zonare z.one clinical ultrasound system using a 4-9-MHz endovaginal transducer. This ultrasound system provides access to radiofrequency (RF) image data for processing and analysis. In addition, a scan of a tissue-mimicking phantom with a known attenuation coefficient was acquired and used as a reference. The same settings and transducer used in the clinical scan were used in the reference scan. Digital data of the beam-formed image were saved in Digital Imaging and Communications in Medicine (DICOM) format on a flash drive and converted to RF data on a personal computer using a Matlab program supplied by Zonare. Attenuation estimates were obtained using an algorithm that was independently validated using tissue-mimicking ultrasonic phantoms. RESULTS: RF data were acquired and analyzed to estimate attenuation of the human pregnant cervix. Regression analysis revealed that attenuation was: a predictor of the interval from ultrasound examination to delivery (beta = 0.43, P = 0.01); not a predictor of gestational age at time of examination (beta = - 0.23, P = 0.15); and not a predictor of cervical length (beta = 0.077, P = 0.65). CONCLUSIONS: Ultrasonic attenuation estimates have the potential to be an early and objective non-invasive method to detect interval between examination and delivery. We hypothesize that a larger sample size and a longitudinal study design will be needed to detect gestational age-associated changes in cervical attenuation.

A new class of organic nitrates: investigations on bioactivation, tolerance and cross-tolerance phenomena.

BACKGROUND AND PURPOSE: The chronic use of organic nitrates is limited by serious side effects including oxidative stress, nitrate tolerance and/or endothelial dysfunction. The side effects and potency of nitroglycerine depend on mitochondrial aldehyde dehydrogenase (ALDH-2). We sought to determine whether this concept can be extended to a new class of organic nitrates with amino moieties (aminoalkyl nitrates). EXPERIMENTAL APPROACH: Vasodilator potency of the organic nitrates, in vitro tolerance and in vivo tolerance (after continuous infusion for 3 days) were assessed in wild-type and ALDH-2 knockout mice by isometric tension studies. Mitochondrial oxidative stress was analysed by L-012-dependent chemiluminescence and protein tyrosine nitration. KEY RESULTS: Aminoethyl nitrate (AEN) showed an almost similar potency to glyceryl trinitrate (GTN), even though it is only a mononitrate. AEN-dependent vasodilatation was mediated by cGMP and nitric oxide. In contrast to triethanolamine trinitrate (TEAN) and GTN, AEN bioactivation did not depend on ALDH-2 and caused no in vitro tolerance. In vivo treatment with TEAN and GTN, but not with AEN, induced cross-tolerance to acetylcholine (ACh)-dependent and GTN-dependent relaxation. Although all nitrates tested induced tolerance to themselves, only TEAN and GTN significantly increased mitochondrial oxidative stress in vitro and in vivo. CONCLUSIONS AND IMPLICATIONS: The present results demonstrate that not all high potency nitrates are bioactivated by ALDH-2 and that high potency of a given nitrate is not necessarily associated with induction of oxidative stress or nitrate tolerance. Obviously, there are distinct pathways for bioactivation of organic nitrates, which for AEN may involve xanthine oxidoreductase rather than P450 enzymes.

Mitochondrial aldehyde dehydrogenase (ALDH-2)--maker of and marker for nitrate tolerance in response to nitroglycerin treatment.

The hemodynamic and anti-ischemic effects of nitroglycerin (GTN) are rapidly blunted as a result of the development of nitrate tolerance. Long-term nitrate treatment also is associated with decreased vascular responsiveness caused by changes in intrinsic mechanisms of the tolerant vasculature itself. According to the oxidative stress concept, increased vascular superoxide and peroxynitrite production as well as an increased sensitivity to vasoconstrictors secondary to activation of protein kinase C as well as vascular NADPH oxidases contribute to the development of tolerance. Recent experimental work has defined new tolerance mechanisms, including inhibition of the enzyme that bioactivates GTN (e.g. mitochondrial aldehyde dehydrogenase [ALDH-2]) and mitochondria as potential sources of reactive oxygen species (ROS). GTN-induced ROS inhibit the bioactivation of GTN by ALDH-2. Both mechanisms impair GTN bioactivation, and now converge at the level of ALDH-2 to support a new theory for GTN tolerance and GTN-induced endothelial dysfunction. The consequences of these processes for GTN downstream targets (e.g. soluble guanylyl cyclase, cyclic guanosine monophosphate-dependent protein kinase) and toxic effects contributing to endothelial dysfunction (e.g. prostacyclin synthase inhibition and NO synthase uncoupling) are discussed. Tolerance and endothelial dysfunction are distinct processes which rely on different sources of ROS and there is good evidence for a crosstalk between these distinct processes. Finally, we will address the question whether ALDH-2 inactivation by nitroglycerin could be a useful marker for clinical nitrate tolerance and discuss the redox-regulation of this enzyme by oxidative stress and dihydrolipoic acid.

New insights into bioactivation of organic nitrates, nitrate tolerance and cross-tolerance.

Organic nitrates still represent a group of very effective anti-ischemic drugs used for the treatment of patients with stable angina, acute myocardial infarction and chronic congestive heart failure. Long-term therapy with organic nitrates, however, results in a rapid development of nitrate tolerance blunting their hemodynamic and antiischemic efficacy. Recent studies revealed that mitochondrial reactive oxygen species (ROS) formation and a subsequent oxidative inactivation of nitrate reductase, the mitochondrial aldehyde dehydrogenase (ALDH-2), play an important role for the development of nitrate and crosstolerance. The present review focuses firstly on the role of ALDH-2 for organic nitrate bioactivation and secondly on the role of oxidative stress in the development of tolerance and cross-tolerance (endothelial dysfunction) in response to various organic nitrates. Finally, we would like to draw the reader's attention to the protective properties of the organic nitrate pentaerithrityl tetranitrate (PETN), which, in contrast to all other organic nitrates, is able to upregulate enzymes with a strong antioxidative capacity thereby preventing tolerance and the development of endothelial dysfunction.

Number of nitrate groups determines reactivity and potency of organic nitrates: a proof of concept study in ALDH-2-/- mice.

BACKGROUND AND PURPOSE: Mitochondrial aldehyde dehydrogenase (ALDH-2) has been shown to provide a pathway for bioactivation of organic nitrates and to be prone to desensitization in response to highly potent, but not to less potent, nitrates. We therefore sought to support the hypothesis that bioactivation by ALDH-2 critically depends on the number of nitrate groups within the nitrovasodilator. EXPERIMENTAL APPROACH: Nitrates with one (PEMN), two (PEDN; GDN), three (PETriN; glyceryl trinitrate, GTN) and four (pentaerithrityl tetranitrate, PETN) nitrate groups were investigated. Vasodilatory potency was measured in isometric tension studies using isolated aortic segments of wild type (WT) and ALDH-2-/- mice. Activity of the cGMP-dependent kinase-I (reflected by levels of phosphorylated VAsodilator Stimulated Phosphoprotein, P-VASP) was quantified by Western blot analysis, mitochondrial dehydrogenase activity by HPLC. Following incubation of isolated mitochondria with PETN, PETriN-chromophore and PEDN, metabolites were quantified using chemiluminescence nitrogen detection and mass spectrometry. KEY RESULTS: Compared to WT, vasorelaxation in response to PETN, PETriN and GTN was attenuated about 10fold in ALDH-2-/- mice, identical to WT vessels preincubated with inhibitors of ALDH-2. Reduced vasodilator potency correlated with reduced P-VASP formation and diminished biotransformation of the tetranitrate- and trinitrate-compounds. None of these findings were observed for PEDN, GDN and PEMN. CONCLUSIONS AND IMPLICATIONS: Our results support the crucial role of ALDH-2 in bioactivating highly reactive nitrates like GTN, PETN and PETriN. ALDH-2-mediated relaxation by organic nitrates therefore depends mainly on the number of nitrate groups. Less potent nitrates like PEDN, GDN and PEMN are apparently biotransformed by other pathways.

Roughness characterization of porous soil with acoustic backscatter.

The use of acoustics to determine the pore properties of soils, such as porosity, permeability, and tortuosity, is well established. A theoretical surface impedance and complex bulk wavenumber was developed by K. Attenborough for porous media that incorporated the soil pore properties as parameters [J. Acoust. Soc. Am. 73, 785-799 (1983)]. Acoustic level difference measurements were used as a noninvasive means of finding the soil pore properties. Acoustic reflection measurements showed that the sound field over porous rough surfaces is modified by the surface impedance and by surface roughness. It is not possible to separate the signal modification due to impedance and the signal modification from roughness scattering in a forward scattering measurement. In order to accurately determine the soil pore properties, the roughness effects must be known independently from the surface impedance. A means of measuring roughness apart from impedance would allow the effects of roughness to be taken out of the level difference measurements. The underwater acoustics community has used acoustic backscatter for many years to examine surface roughness. The feasibility of adapting these acoustic backscatter techniques to outdoor porous soil surfaces is examined.

Effects of in vivo nitroglycerin treatment on activity and expression of the guanylyl cyclase and cGMP-dependent protein kinase and their downstream target vasodilator-stimulated phosphoprotein in aorta.

BACKGROUND: Chronic in vivo treatment with nitroglycerin (NTG) induces tolerance to nitrates and cross-tolerance to nitrovasodilators and endothelium-derived nitric oxide (NO). We previously identified increased vascular superoxide formation and reduced NO bioavailability as one causal mechanism. It is still controversial whether intracellular downstream signaling to nitrovasodilator-derived NO is affected as well. METHODS AND RESULTS: We therefore studied the effects of 3-day NTG treatment of rats and rabbits on activity and expression of the immediate NO target soluble guanylyl cyclase (sGC) and on the cGMP-activated protein kinase I (cGK-I). Tolerance was induced either by chronic NTG infusion via osmotic minipumps (rats) or by NTG patches (rabbits). Western blot analysis, semiquantitative reverse transcription-polymerase chain reaction, and Northern blot analysis revealed significant and comparable increases in the expression of sGC alpha(1) and beta(1) subunit protein and mRNA. Studies with the oxidative fluorescent dye hydroethidine revealed an increase in superoxide in the endothelium and smooth muscle. Stimulation with NADH increased superoxide signals in both layers. Although cGK-I expression in response to low-dose NTG was not changed, a strong reduction in vasodilator-stimulated phosphoprotein (VASP) serine239 phosphorylation (specific substrate of cGK-I) was observed in tolerant tissue from rats and rabbits. Concomitant in vivo and in vitro treatment with vitamin C improved tolerance, reduced oxidative stress, and improved P-VASP. CONCLUSIONS: We therefore conclude that increased expression of sGC in the setting of tolerance reflects a chronic inhibition rather than an induction of the sGC-cGK-I pathway and may be mediated at least in part by increased vascular superoxide.

Antioxidants and endothelial dysfunction in hyperlipidemia.

Endothelial function is abnormal in a variety of diseased states such as hypercholesterolemia and atherosclerosis. This may be secondary to decreased synthesis of nitric oxide (NO) and/or increased degradation of NO due to interaction with superoxide anions. More recent experimental observations demonstrate increased production of superoxide in hyperlipidemia, suggesting that endothelial dysfunction in these states is in part secondary to increased NO metabolism. Enzymes proposed to be involved in increased superoxide production may include xanthine oxidase, the NO synthase, and the NAD(P)H oxidase. Superoxide rapidly reacts with NO to form peroxynitrite (ONOO-), a highly reactive intermediate with cytotoxic properties. Despite experimental evidence for the oxidative stress concept in causing endothelial dysfunction, the results of recent randomized trials to test the influence of antioxidants on coronary event rates and prognosis in patients with coronary artery disease were very disappointing. In all of these studies the use of vitamins such as vitamin E failed to improve the prognosis. In contrast, treatment with angiotensin converting enzyme inhibitors or cholesterol- lowering drugs improved endothelial dysfunction, prevented the activation of superoxide-producing enzymes in cholesterol-fed animals, reduced coronary event rates, and improved prognosis in patients with coronary artery disease. Therefore, inhibition of superoxide production at the enzymatic level rather than symptomatic superoxide scavenging may be the better choice of treatment.

Mechanisms underlying endothelial dysfunction in diabetes mellitus.

Incubation of endothelial cells in vitro with high concentrations of glucose activates protein kinase C (PKC) and increases nitric oxide synthase (NOS III) gene expression as well as superoxide production. The underlying mechanisms remain unknown. To address this issue in an in vivo model, diabetes was induced with streptozotocin in rats. Streptozotocin treatment led to endothelial dysfunction and increased vascular superoxide production, as assessed by lucigenin- and coelenterazine-derived chemiluminescence. The bioavailability of vascular nitric oxide (as measured by electron spin resonance) was reduced in diabetic aortas, although expression of endothelial NOS III (mRNA and protein) was markedly increased. NOS inhibition with N:(G)-nitro-L-arginine increased superoxide levels in control vessels but reduced them in diabetic vessels, identifying NOS as a superoxide source. Similarly, we found an activation of the NADPH oxidase and a 7-fold increase in gp91(phox) mRNA in diabetic vessels. In vitro PKC inhibition with chelerythrine reduced vascular superoxide in diabetic vessels, whereas it had no effect on superoxide levels in normal vessels. In vivo PKC inhibition with N:-benzoyl-staurosporine did not affect glucose levels in diabetic rats but prevented NOS III gene upregulation and NOS-mediated superoxide production, thereby restoring vascular nitric oxide bioavailability and endothelial function. The reduction of superoxide in vitro by chelerythrine and the normalization of NOS III gene expression and reduction of superoxide in vivo by N:-benzoyl-staurosporine point to a decisive role of PKC in mediating these phenomena and suggest a therapeutic potential of PKC inhibitors in the prevention or treatment of vascular complications of diabetes mellitus. The full text of this article is available at http://www.circresaha.org.

Vasodilator-stimulated phosphoprotein serine 239 phosphorylation as a sensitive monitor of defective nitric oxide/cGMP signaling and endothelial dysfunction.

Studies with cGMP-dependent protein kinase I (cGK-I)-deficient human cells and mice demonstrated that cGK-I ablation completely disrupts the NO/cGMP pathway in vascular tissue, which indicates a key role of this protein kinase as a mediator of the NO/cGMP action. Analysis of the vasodilator-stimulated phosphoprotein phosphorylated at serine 239 (P-VASP) is a useful tool to monitor cGK-I activation in platelets and cultured endothelial and smooth muscle cells. Therefore, we investigated whether endothelial dysfunction and/or vascular NO bioavailability is reflected by decreased vessel wall P-VASP and whether improvement of endothelial dysfunction restores this P-VASP. Incubation of aortic tissue from New Zealand White Rabbits with the NOS inhibitor N:(G)-nitro-Ld-arginine and endothelial removal strikingly reduced P-VASP. Oxidative stress induced by inhibition of CuZn superoxide dismutase increased superoxide and decreased P-VASP. Endothelial dysfunction in hyperlipidemic Watanabe rabbits (WHHL) was associated with increased vascular superoxide and with decreased P-VASP. Treatment of WHHL with AT(1) receptor blockade improved endothelial dysfunction, reduced vascular superoxide, increased vascular NO bioavailability, and increased P-VASP. Therefore, the level of vessel P-VASP closely follows changes in endothelial function and vascular oxidative stress. P-VASP is suggested to represent a novel biochemical marker for monitoring the NO-stimulated sGC/cGK-I pathway and endothelial integrity in vascular tissue.

Spin trapping of vascular nitric oxide using colloid Fe(II)-diethyldithiocarbamate.

Currently available EPR spin-trapping techniques are not sensitive enough for quantification of basal vascular nitric oxide (NO) production from isolated vessels. Here we demonstrate that this goal can be achieved by the use of colloid Fe(DETC)(2). Rabbit aortic or venous strips incubated with 250 microM colloid Fe(DETC)(2) exhibited a linear increase in tissue-associated NO-Fe(DETC)(2) EPR signal during 1 h. Removal of endothelium or addition of 3 mM N(G)-nitro-l-arginine methyl ester (L-NAME) inhibited the signal. The basal NO production was estimated as 5.9 +/- 0.5 and 8.3 +/- 2.1 pmol/min/cm(2) in thoracic aorta and vena cava, respectively. Adding sodium nitrite (10 microM) or xanthine/xanthine oxidase in the incubation medium did not modify the intensity of the basal NO-Fe(DETC)(2) EPR signal. Reducing agents were not required with this method and superoxide dismutase activity was unchanged by the Fe(DETC)(2) complex. We conclude that colloid Fe(DETC)(2) may be a useful tool for direct detection of low amounts of NO in vascular tissue.

Effects of a nitrate-free interval on tolerance, vasoconstrictor sensitivity and vascular superoxide production.

OBJECTIVES: In the present study, we tested whether a nitrate-free interval is able to prevent increases in vascular superoxide (O2*-) and the development of hypersensitivity to vasoconstrictors and whether this may result in restoration of vascular nitroglycerin (NTG) sensitivity. BACKGROUND: Intermittent NTG-patch treatment (12 h patch on/patch-off) has been shown to increase ischemic periods in patients with stable coronary arteries, suggesting a rebound-like situation during the patch-off period. Recently, we demonstrated that long-term treatment with NTG induces tolerance, which was in part related to increases in vascular O2*- and increased vasoconstrictor sensitivity. METHODS: New Zealand white rabbits received a continuous application of NTG patches (0.4 mg/h) or an intermittent application of NTG patches (12 h patch on, 12 h patch off) for three days. Isometric tension studies were performed with aortic rings, and vascular O2*- was estimated using lucigenin-derived chemiluminescence (5 micromol/liter). Expression of the copper/zinc (Cu/Zn) superoxide dismutase (SOD) was assessed by Western blotting, and SOD activity was measured by autooxidation of 6-hydroxydopamine. RESULTS: Continuous treatment with NTG caused tolerance to NTG, cross-tolerance to the endothelium-dependent vasodilator acetylcholine, increased vascular O2*-, reduced Cu/Zn SOD expression and increased sensitivity to vasoconstrictors such as phenylephrine, serotonin and angiotensin II. On/off treatment with NTG improved tolerance, corrected endothelial dysfunction and decreased vascular O2*-. In addition the reduction in SOD expression was less pronounced, whereas increases in the sensitivity to vasoconstrictors such as phenylephrine and serotonin remained nearly unchanged. CONCLUSIONS: Enhanced vasoconstrictor sensitivity may explain, at least in part, the rebound phenomena observed in patients during a 12-h NTG patch-off period.

Effects of long-term nitroglycerin treatment on endothelial nitric oxide synthase (NOS III) gene expression, NOS III-mediated superoxide production, and vascular NO bioavailability.

Long-term nitroglycerin (NTG) treatment has been shown to be associated with cross-tolerance to endothelium-dependent vasodilators. It may involve increased production of reactive oxygen species (such as superoxide, O(2)(.-)) that rapidly inactivate the nitric oxide (NO) released from the endothelial cells. It remains to be elucidated, however, whether long-term treatment with NTG alters the activity and expression of the endothelial NO synthase (NOS III) and whether this enzyme can contribute to O(2)(.-) formation. We studied the influence of long-term NTG treatment on the expression of NOS III as assessed by RNase protection assay and Western blot. Tolerance was measured ex vivo in organ chamber experiments with rat aortic rings. O(2)(.-) and NO formation were quantified using lucigenin- and Cypridina luciferin analog-enhanced chemiluminescence as well as electron spin resonance (ESR) spectroscopy. Treatment of Wistar rats with NTG (Alzet osmotic minipumps, NTG concentration 10 microg x kg(-1) x min(-1)) for 3 days caused marked tolerance, cross-tolerance to the endothelium-dependent vasodilator acetylcholine, and a significant increase in O(2)(.-)-induced chemiluminescence. Tolerance was associated with a significant increase in NOS III mRNA to 236+/-28% and NOS III protein to 239+/-17%. In control vessels, the NOS inhibitor N(G)-nitro-L-arginine (L-NNA) increased the O(2)(.-)-mediated chemiluminescence, indicating that basal production of endothelium-derived NO depresses the baseline chemiluminescence signal. In the setting of tolerance, however, L-NNA decreased steady-state O(2)(.-) levels, indicating the involvement of NOS III in O(2)(.-) formation. Likewise, A23187-induced, NOS III-mediated O(2)(.-) production was more pronounced in tolerant than in control vessels. Vascular NO bioavailability as assessed with ESR spectroscopy using iron-thiocarbamate as a trap for NO was significantly reduced in tolerant vessels. Pretreatment of tolerant tissue in vitro with the protein kinase C (PKC) inhibitors reduced basal and stimulated NOS III-mediated O(2)(.-) production and partially reversed vascular tolerance. These findings suggest that NTG treatment increases the expression of a dysfunctional NOS III gene, leading to increased formation of O(2)(.-) and decreased vascular NO bioavailability. Normalization of NOS III-mediated O(2)(. -) production and improvement of tolerance with PKC inhibition suggests an important role for PKC isoforms in mediating vascular dysfunction caused by long-term NTG treatment.