Isolation of nitric oxide synthase from human platelets.

We are reporting a distinct constitutive isoform of nitric oxide synthase that has been purified to homogeneity from human platelet cytosolic fractions. Purification involved ultra centrifugation at 100,000 x g followed by two sequential affinity chromatography procedures: adenosine 2',5'-bisphosphate (2',5'-ADP)-Sepharose and calmodulin Sepharose 4B. Purified enzyme appeared as a single band (approximately 80 kDa) under denaturing condition (SDS-PAGE). The native enzyme appears to be dimeric, since its molecular weight estimated by gel filtration was approximately 150 kDa. Enzyme activity was dependent on L-arginine, NADPH and (6R)-5,6,7,8-tetrahydro-L-biopterine. Partially purified platelet NOS (100,000 x g supernatant) activity was sensitive to calmodulin antagonists and to the N omega-Monomethyl-L-arginine, a substrate analog of L-arginine.

Identification of the site of non-enzymatic glycation of glutathione peroxidase: rationalization of the glycation-related catalytic alterations on the basis of three-dimensional protein structure.

Bovine erythrocyte glutathione peroxidase has been glycated in vitro by incubation in 0.05 M glucose at pH 7.4. Upon glycation the estimated KM for t-butylhydroperoxide reduction increased by approx. 3-fold in comparison to non-glycated glutathione peroxidase. The glycated protein fraction was stabilized by NaBH4 reduction and subjected to tryptic cleavage. Affinity chromatography of the tryptic digest on m-aminophenylboronate-Agarose resulted in the isolation of a single glycated peptide. The peptide was identified as T94-K117 by amino-acid composition comparison to the published amino-acid sequence for this enzyme. The glycation site has been identified as the epsilon-NH2 group of K110. Examination of the three-dimensional structure of bovine erythrocyte glutathione peroxidase indicates that K110 lies on the surface of the protein approximately 15 A away from the active site selenocysteine (SEC 45). Modeling studies indicate that K110 can communicate via H-bonded interactions with the alpha-helix containing the active site residues (SEC-45 and R50). The observed elevation of KM upon glycation of bovine glutathione peroxidase is discussed in terms of the disruption of the long range H-bonded interaction.

N-dansyl-S-nitrosohomocysteine a fluorescent probe for intracellular thiols and S-nitrosothiols.

The fluorescence emission spectrum of N-dansyl-S-nitrosohomocysteine was enhanced approximately 8-fold upon removal of the NO group either by photolysis or by transnitrosation with free thiols like glutathione. The fluorescence enhancement was reversible in that it could be quenched in the presence of excess S-nitrosoglutathione. Attempts were then made to utilize N-dansyl-S-nitrosohomocysteine as an intracellular probe of thiols/S-nitrosothiols. Fluorescence microscopy of fibroblasts in culture indicated that intracellular N-dansyl-S-nitrosohomocysteine levels reached a maximum within 5 min. N-Dansyl-S-nitrosohomocysteine fluorescence was directly proportional to intracellular GSH levels, directly determined with HPLC. N-Dansyl-S-nitrosohomocysteine preloaded cells were also sensitive to S-nitrosoglutathione uptake as the intracellular fluorescence decreased as a function of time upon exposure to extracellular S-nitrosoglutathione.

Evidence for peroxynitrite formation during S-nitrosoglutathione photolysis in air saturated solutions.

Flash photolysis of S-nitrosoglutathione (GSNO) in aerated solutions at pH 10 gave rise to an absorption with a maximum around 310-320 nm. This peak is spectrally similar to that displayed by ONOO-. The decay kinetics of this absorption was compared to that of authentic ONOO-, generated independently. An excellent correlation was obtained. Further proof of ONOO- generation was provided by HPLC studies showing the production of 3-nitrotyrosine on irradiation of GSNO in the presence of tyrosine at pH 7.4. In addition, the nitration yield was increased approximately 5-fold in the presence of bicarbonate and totally eliminated with DMPO, indicating the requirement of a radical intermediate for peroxynitrite production during S-nitrosothiol photolysis.

Cytotoxic activities of Mannich bases of chalcones and related compounds.

Various Mannich bases of chalcones and related compounds displayed significant cytotoxicity toward murine P388 and L1210 leukemia cells as well as a number of human tumor cell lines. The most promising lead molecule was 21 that had the highest activity toward L1210 and human tumor cells. In addition, 21 exerted preferential toxicity to human tumor lines compared to transformed human T-lymphocytes. Other compounds of interest were 38, with a huge differential in cytotoxicity between P388 and L1210 cells, and 42, with a high therapeutic index when cytotoxicity to P388 cells and Molt 4/C8 T-lymphocytes were compared. In general, the Mannich bases were more cytotoxic than the corresponding chalcones toward L1210 but not P388 cells. A ClusCor analysis of the data obtained from the in vitro human tumor screen revealed that the mode of action of certain groups of compounds was similar. For some groups of compounds, cytotoxicity was correlated with the sigma, pi, or molar refractivity constants in the aryl ring attached to the olefinic group. In addition, the IC50 values in all three screens correlated with the redox potentials of a number of Mannich bases. X-ray crystallography and molecular modeling of representative compounds revealed various structural features which were considered to contribute to cytotoxicity. While a representative compound 15 was stable and unreactive toward glutathione (GSH) in buffer, the Mannich bases 15, 18, and 21 reacted with GSH in the presence of the pi isozyme of glutathione S-transferase, suggesting that thiol alkylation may be one mechanism by which cytotoxicity was exerted in vitro. Representative compounds were shown to be nonmutagenic in an intrachromosomal recombination assay in yeast, devoid of antimicrobial properties and possessing anticonvulsant and neurotoxic properties. Thus Mannich bases of chalcones represent a new group of cytotoxic agents of which 21 in particular serves as an useful prototypic molecule.

S-nitrosoglutathione photolysis as a novel therapy for antifibrosis in filtration surgery.

PURPOSE: To determine whether a novel peroxynitrite-based photosensitizer S-nitrosoglutathione (GSNO) can produce specific in vitro light-induced cell death of both standard animal lung and human Tenon's capsule (TC) fibroblasts and to compare this effect with that produced by the established photodynamic porphyrin precursor 5-aminolevulinic acid (ALA). METHODS: V79-4 Chinese hamster lung and human TC fibroblasts were established in tissue culture. GSNO, together with its radioactive tritiated and fluorescent dansylated derivatives, were synthesized. The labeled molecules were prepared to determine the time course of uptake into the fibroblasts. Uptake was monitored by scintillation counting for the tritiated GSNO and confocal fluorescence microscopy for the dansylated GSNO. The uptake of ALA and biosynthesis of its photosensitive product were determined by fluorescence emission spectroscopy of a separate set of fibroblasts. Once uptake was established, both cell lines were incubated with varying concentrations of GSNO or ALA as a function of time (0, 4, or 24 hours) before light exposure (200 msec pulsed visible light, 0.068 W per pulse, for 10 minutes at a distance of 10 cm). After 10 minutes of irradiation, the cells were washed and exposed to fresh tissue culture medium. The effect of the treatment was determined 24 hours later by measuring cell viability. RESULTS: A 2-minute drug treatment time (0 hours incubation) with GSNO, followed by 10 minutes of irradiation, resulted in approximately 78% of fibroblast cell death at the lowest concentration of GSNO used compared with the control, which was exposed to light, but no GSNO. The higher concentrations of GSNO, or longer drug treatment times before irradiation, did not statistically increase cell death. Maximal cell death was thus obtained using the lowest GSNO concentration (50 mM) and drug treatment time (2 minutes). In contrast, the well-established photosensitizer ALA killed only approximately 4% of cells at the lowest concentration and drug treatment time tested. At drug treatment times of 4 hours and less, increased concentrations of ALA did not produce cell death of more statistical significance. It was not until 24 hours of drug treatment that comparable amounts of cell death were produced by ALA and GSNO. In all experiments similar results were obtained with the animal lung and human TC fibroblasts, suggesting that the source of the fibroblast had no effect on the outcome. The differences in treatment effects between GSNO and ALA were statistically significant under all conditions tested. CONCLUSIONS: GSNO is able to cause light-specific cell death of human TC fibroblasts at drug treatment times (2 minutes) and irradiation times (10 minutes) that would be compatible with its use in glaucoma filtering surgery. This in vitro performance was superior to that of the well-established photosensitizer ALA, which required treatment times longer than 4 hours to approach the light-specific cell death produced by only 2 minutes of GSNO treatment.

Atorvastatin increases ecNOS levels in human platelets of hyperlipidemic subjects.

BACKGROUND: The purpose of this study was to probe the pleiotrophic effects of Atorvastatin on intraplatelet-nitric oxide metabolism. METHODS AND RESULTS: Hyperlipidemic subjects (n = 19) were treated for 1 month (following a 3-week washout) with either Atorvastatin or placebo in a double-blinded randomized (n = 2, crossover), placebo-controlled study. Changes in the levels of intraplatelet nitric oxide synthase, nitrotyrosine were correlated with cholesterol, LDL-C, HDL-C and triglyceride levels. These studies indicate that with atrovastatin ecNOS levels increased on average by approximately approximately 1.7-fold (paired t-test p = 0.009). Interestingly, levels of nitrotyrosylated platelet proteins, an indication of peroxynitrite damage, decreased as ecNOS levels increased in presence of the drug (paired t-test p = 0.33). Atorvastatin, at 10 mg per day, lowered cholesterol and LDL-C levels in all patients with the average lowering of approximately 21% and approximately 17% respectively. The effect on HDL was not significant whilst triglyceride levels were lowered by an average of approximately 18%. CONCLUSIONS: This study adds to the volume of evidence that statins have beneficial effects other than lipid lowering. Here, Atorvastatin is shown to significantly elevate intraplatelet ecNOS levels in hyperlipidemic subjects without affecting iNOS expression. The net result of this would be the elevation of NO production which would promote platelet deaggregation and vasodilation.

Glutathione levels during thermal induction of the yeast-to-mycelial transition in Candida albicans.

Glutathione (GSH) levels were directly monitored by reverse phase HPLC during the thermal yeast-to-mycelial induction of Candida albicans. The GSH levels decreased approximately 100-fold within 120 min which corresponded to the time of maximal yeast-to-mold conversion. The yeast to mold conversion was inhibited by 1-p-chlorophenyl-4,4-dimethyl-5-diethylamino-1-penten-3-one (CDDP), a thiol-specific alkylator, which prevented the decline in GSH levels. These results are discussed with respect to the potential involvement of intracellular GSH levels in regulation of the yeast-to-mold dimorphism in Candida albicans.

Diabetes-induced alterations in platelet metabolism.

OBJECTIVES: This review summarizes the recent findings on some aspects of platelet metabolism that appear to be affected as a consequence of diabetes mellitus. The metabolites include glutathione, L-Arginine/nitric oxide, as well as the ATP-dependent exchange of Na+/K+ and Ca2+. CONCLUSIONS: Several aspects of platelet metabolism are altered in diabetics. These metabolic events give rise to a platelet that has less antioxidants, and higher levels of peroxides. The direct consequence of this is the overproduction platelet agonists. In addition, there is evidence for altered Ca2+ and Na+ transport across the plasma membrane. Recent evidence indicates that plasma ATPases in diabetic platelets are not damaged instead their activities are likely to be modulated by oxidized LDL. Finally, platelet inhibitory mechanisms regulated by NO appear to be perturbed in the diabetes disease-state. The combined production of NO and superoxide by NOS isoforms in the platelet could be a major contributory factor to platelet pathogenesis in diabetes mellitus.

ELISA for in vivo assessment of nonenzymatically glycated platelet glutathione peroxidase.

Using a combination of boronate affinity chromatography and ELISA methodology, a simple procedure was devised to selectively determine the in vivo glycated state of the platelet glutathione peroxidase (GSH-Px) from normal and diabetic subjects. The mean total GSH-Px levels in the normal (n = 14) and diabetic (n = 18) platelets were 1167 +/- 97 and 1007 +/- 73 ng/mg protein, respectively. The mean percentage glycated GSH-Px in the normal and diabetic platelets were 5.79 +/- 0.72% and 11.68 +/- 0.95%, respectively. When the percentage glycated GSH-Px was compared with the fructosamine values, a correlation coefficient of 0.71 was obtained. This indicates that the glycation status of platelet GSH-Px can be utilized as a sensitive, short-term index of plasma glucose levels.

S-nitrosoglutathione/glutathione disulphide/Cu2+-dependent stimulation of L-arginine transport in human platelets.

In this study, we have examined the effects of authentic nitric oxide (NO), NO+ (NOBF4), glutathione (GSH), glutathione disulphide (GSSG), and S-nitrosoglutathione (GSNO) in the presence and absence of Cu2+, which thermally releases NO from S-nitrosothiols on the transport of L-arginine into the human platelet. The K(M,apparent) was unaffected by NO, NO+, GSH, and GSNO. However, Cu2+ lowered K(M,apparent) by approximately 2.85-fold. Cu2+-dependent lowering of K(M,apparent) was also observed, albeit to a smaller extent when this ion was mixed with GSH (approximately 1.9-fold lower) and GSNO (approximately 2.0-fold). GSSG also lowered K(M,apparent) by approximately 1.5-fold. The Vmax,apparent of L-arginine uptake was unaffected by NO, NO+, GSH, and Cu2+. Vmax,apparent was stimulated by to the largest extent by GSNO (approximately 2.28-fold) and GSNO plus Cu2+ (approximately 2.7-fold). GSSG and GSH plus Cu2+ also increased Vmax,apparent by approximately 1.9-fold. When these parameters are expressed in terms of transport efficiency (Vmax/K(M)) the largest effect of nearly 4.7-fold (over controls) was obtained by a combination of GSNO plus Cu2+. These results suggest that platelet L-Arg transport is not affected either by NO or NO+ but by a thiol-disulphide exchange reactions on the platelet L-Arg transporter, brought about by GSNO and GSSG. Based on these results, a GSNO/GSSG/Cu2+ dependent regulatory mechanism for the uptake of L-arginine in human platelets has been proposed.

Glutathione metabolic enzyme activities in diabetic platelets as a function of glycemic control.

Type 1 diabetic subjects categorized on the basis of the glycated haemoglobin content of their blood (low less than 7%; medium, greater than 7% and less than 11%; high, greater than 11%) were analyzed for total intraplatelet GSH as well as for the steady-state kinetic parameters (apparent KM and apparent Vmax) of some glutathione metabolic enzymes including glutathione reductase, glutathione peroxidase, gamma-glutamyltrans-peptidase and glutathione-S-transferase. This study indicates that intraplatelet GSH content of subjects with low glycated-haemoglobin is approximately 2-fold higher than those with medium glycated-haemoglobin. There was no further decrease in intraplatelet-GSH in subjects with high glycated-haemoglobin. The kinetic parameters of the platelet-enzymes studied (glutathione reductase, gamma-glutamyltranspeptidase and glutathione-S-transferase) were essentially independent of the glycation state of the subject. However, the apparent KM of glutathione peroxidase was approximately 4-fold higher in the subjects with high glycated-haemoglobin, in comparison to low subjects. This decrease in affinity could possibly result from the susceptibility of this enzyme to non-enzymatic glucosylation as purified samples of glutathione peroxidase incubated in vitro with glucose showed similar increases in apparent KM. These results are discussed in terms of the potential contribution of glutathione peroxidase impairment, to the hyperaggregability of the diabetic platelet.

Visible light photochemical release of nitric oxide from S-nitrosoglutathione: potential photochemotherapeutic applications.

Some aspects of the physiological role of NO may be mediated by stable NO-carriers such as S-nitrosoglutathione and related S-nitrosothiols. In this report we show that irradiation of S-nitrosoglutathione at either absorption band (lambda max = 340 nm or 545 nm) results in the release of nitric oxide. Photolysis of S-nitrosoglutathione at 545 nm exhibited a quantum yield of 0.056 +/- 0.002 and was best approximated by a first-order process with kobs = 4.9 x 10(-7) +/- 0.3 x 10(-7) s-1. The photolytic release of NO from S-nitrosoglutathione resulted in an enhanced cytotoxic effect of S-nitrosoglutathione on HL-60 leukemia cells. That the cytotoxic effect of S-nitrosoglutathione was diminished by the addition of oxyhemoglobin strongly suggests that NO is the cytotoxic species. The finding that NO can be readily liberated from S-nitrosoglutathione by visible radiation indicates that the photochemical properties of this compound in the visible spectrum must be considered in order to obtain meaningful data as to its physiological role and the S-nitrosoglutathione and related compounds may find use as photochemotherapeutic agents.

A photo-activated, protein-based, NO/H2O2 generating system with tumoricidal activity composed of the nitric oxide derivative of apo-metallothionein (thionein-NO) and glucose oxidase.

The S-nitroso derivative of apo-metallothionein (thionein) was prepared by transnitrosation with S-nitrosoglutathione. The thionein-NO thus formed has an absorption maximum at 334 nm. Light-induced NO release from thionein-NO was demonstrated by flash photolysis. This system produces peroxynitrite at neutral pH as evidenced by nitrotyrosine formation. The cytotoxic potential of this protein-based, light-activated NO/H2O2 generating system was demonstrated by exposing human colon adenocarcinoma cells (SW 948) in culture to thionein-NO and glucose oxidase in the presence and absence of light. The cell density of the samples, 72 h subsequent to receiving 1 h of light exposure, decreased by approximately 98%, relative to controls. In comparison, cell density of the samples that were incubated in the presence of catalase and did not receive light treatment, decreased by only approximately 22% after 72 h.

Effects of therapeutic touch on biochemical and mood indicators in women.

Previous research has shown therapeutic touch (TT) to be effective in reducing anxiety and discomfort and promoting relaxation. The present investigation experimentally evaluated the effects of TT on biochemical indicators and moods in a sample of 41 healthy female volunteers. Participants were randomly assigned to either an experimental group who received TT or to a control group who did not receive TT. Pretest and posttest urine samples were collected, and personality and mood inventories were administered across three consecutive monthly sessions. Results indicated that mood disturbance in the experimental group decreased significantly over the course of the three sessions, while the control group increased in mood disturbance over time. Specifically, experimental group participants showed significant reductions in tension, confusion, and anxiety and a significant increase in vigor across sessions. Analyses of the biochemical data indicated that TT produced a significant decrease in levels of nitric oxide in the experimental group by the third TT session. The results of the present investigation have important implications for reducing symptom distress in cancer patients undergoing chemotherapy.

Transient activation of calcineurin during thiol modification.

Thiol titrations of bovine brain calcineurin, phosphatase with Ellman's reagent revealed the presence of 5 exposed sulfhydryl groups on the native protein, and 10 sulfhydryl groups on the denatured protein. Attempts were made to identify the location of the free thiols within the catalytic and regulatory domains of the enzyme. Our data indicates that free sulfhydryl groups are absent from the vicinity of the Mg2+ and calmodulin binding sites as well as from the active site of the enzyme. However, the fact that the number of free thiols decreased in the presence of Ca2+ and Mn2+, to 4 and 2 respectively, possibly indicates that either free thiols are at or near these domains or become inaccessible as a consequence of conformational changes induced by the metal ions. The Ca2+ and Ca2+/Mg2+ stimulated activities of calcineurin were monitored during modification with Ellman's reagent, iodoacetate and iodoacetamide. Upon modification of 1-2 of the free thiols the activity of the enzyme increased 1.3 to 10.5-fold depending on the thiol reagent and the stimulatory metal ions employed. Modification of the remainder of the free thiols resulted in a decrease in activity. These results suggest that 1-2 thiols are essential for the full expression of calcineurin activity.

Selective adsorption of 2'-O-anthraniloyl-AMP on DEAE-Sephadex: the basis of a direct, fluorescent assay for cyclic nucleotide phosphodiesterase.

The fluorescent, 2'-O-anthraniloyl derivative of AMP was selectively absorbed onto DEAE-Sephadex in the presence of zirconyl chloride in citrate buffer. Under these conditions 2'-O-anthraniloyl-cAMP was eluted from the column. The selective adsorption of the AMP derivative onto DEAE-Sephadex, in the presence of zirconyl chloride, was adapted to the direct discontinuous assay of cyclic nucleotide phosphodiesterase. In this assay the enzyme is incubated for 4 min with 2'-O-anthraniloyl-cAMP; after quenching of the reaction by boiling, zirconyl chloride is added and the product (2'-O-anthraniloyl-AMP) is separated from the substrate on a column (0.6 ml) of DEAE-Sephadex. 2'-O-Anthraniloyl-AMP is then eluted with NaCl (2 M) and quantitated spectrofluorometrically. Under the conditions employed, 2'-O-anthraniloyl-AMP concentrations as low as 0.1 nmol can be detected. In the present study, this assay has been used to estimate Km and Vmax values for 2'-O-anthraniloyl-cAMP hydrolysis catalyzed by highly purified, as well as crude, preparations of cyclic nucleotide phosphodiesterase from bovine brain.