calcimycin and 3-nitrotyrosine

We have shown that pulmonary nanoparticle exposure impairs endothelium dependent dilation in systemic arterioles. However, the mechanism(s) through which this effect occurs is/are unclear. The purpose of this study was to identify alterations in the production of reactive species and endogenous nitric oxide (NO) after nanoparticle exposure, and determine the relative contribution of hemoproteins and oxidative enzymes in this process. Sprague-Dawley rats were exposed to fine TiO2 (primary particle diameter approximately 1 microm) and TiO2 nanoparticles (primary particle diameter approximately 21 nm) via aerosol inhalation at depositions of 4-90 microg per rat. As in previous intravital experiments in the spinotrapezius muscle, dose-dependent arteriolar dilations were produced by intraluminal infusions of the calcium ionophore A23187. Nanoparticle exposure robustly attenuated these endothelium-dependent responses. However, this attenuation was not due to altered microvascular smooth muscle NO sensitivity because nanoparticle exposure did not alter arteriolar dilations in response to local sodium nitroprusside iontophoresis. Nanoparticle exposure significantly increased microvascular oxidative stress by approximately 60%, and also elevated nitrosative stress fourfold. These reactive stresses coincided with a decreased NO production in a particle deposition dose-dependent manner. Radical scavenging, or inhibition of either myeloperoxidase or nicotinamide adenine dinucleotide phosphate oxidase (reduced) oxidase partially restored NO production as well as normal microvascular function. These results indicate that in conjunction with microvascular dysfunction, nanoparticle exposure also decreases NO bioavailability through at least two functionally distinct mechanisms that may mutually increase local reactive species.

Topics: Animals; Calcimycin; Capillaries; Dose-Response Relationship, Drug; Endothelium, Vascular; Inhalation Exposure; Lung; Male; Nanoparticles; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Oxidative Stress; Particle Size; Rats; Rats, Sprague-Dawley; Signal Transduction; Titanium; Tyrosine; Vasodilation

The objective was to test the hypothesis that dietary copper inhibits atherosclerosis by inducing superoxide dismutase (SOD) and potentiating nitric oxide (NO). New Zealand White rabbits were fed either a cholesterol diet (n = 8) or a cholesterol diet containing 0.02% copper acetate (n = 8) for 13 weeks. We found that the intimal area was significantly smaller in the animals supplemented with copper (P < 0.005), although integrated plasma cholesterol levels were not significantly different. This was associated with a significant increase in aortic copper content (P < 0.05), SOD activity (P < 0.05) and Cu/Zn SOD mRNA (P < 0.05) and a significant decrease in nitrotyrosine content (P < 0.05). Furthermore, there was a positive correlation between aortic copper content and SOD activity (P < 0.005, R(2) = 0.83) and a negative correlation between aortic superoxide dimutase activity and nitrotyrosine content (P < 0.005, R(2) = 0.93). In organ bath experiments, the relaxation of precontracted carotid artery rings to calcium ionophore was greater in animals supplemented with copper. No difference in response to sodium nitroprusside was observed. These data suggest that in the cholesterol-fed rabbit, copper supplements inhibit the progression of atherosclerosis by increasing SOD expression, thereby reducing the interaction of NO with superoxide, and hence potentiating NO-mediated pathways that may protect against atherosclerosis.

Topics: Animals; Aorta, Thoracic; Arteriosclerosis; Calcimycin; Carotid Arteries; Cholesterol; Copper; Dietary Supplements; Ionophores; Muscle, Smooth; Nitric Oxide; Oxidative Stress; Rabbits; Superoxide Dismutase; Tyrosine

Tyrosine nitration is a widely used marker of peroxynitrite (ONOO-) produced from the reaction of nitric oxide (NO.) with superoxide (O2(.-)). Since human spermatozoa are able to produce both NO. and O2(.-) during capacitation in vitro, we investigated whether spontaneous tyrosine nitration of proteins occurs in human spermatozoa and evaluated the physiological effects of peroxynitrite on sperm function. We report here that human spermatozoa, incubated for 8 h under conditions conducive to capacitation, display a reproducible pattern of protein tyrosine nitration. Several proteins with mol. wt of 105-14 kDa become increasingly tyrosine-nitrated after 15 min incubation and then minimal changes are observed. Treatment of capacitated spermatozoa with human follicular fluid or calcium ionophore causes an increase of the nitrotyrosine content of proteins at the mol. wt of 85 kDa. Moreover, exposure of spermatozoa to ONOO- (2.5-50 micromol/l) increases motility and primes spermatozoa to respond earlier to human follicular fluid. ONOO- also increases protein tyrosine nitration and phosphorylation in a concentration-dependent manner. Taken together, these results demonstrate that tyrosine nitration of sperm proteins occurs in capacitated human spermatozoa, and that low concentrations of ONOO- modulate sperm functions, emphasizing the concept that capacitation is part of an oxidative process.

Topics: Calcimycin; Female; Follicular Fluid; Humans; Ionophores; Male; Nitric Oxide; Peroxynitrous Acid; Phosphorylation; Proteins; Sperm Capacitation; Sperm Motility; Spermatozoa; Superoxides; Tyrosine

Hyperhomocysteinemia (HHCy) is an independent and graded cardiovascular risk factor. HHCy is prevalent in patients with chronic renal failure, contributing to the increased mortality rate. Controversy exists as to the effects of HHCy on nitric oxide (NO) production: it has been shown that HHCy both increases and suppresses it. We addressed this problem by using amperometric electrochemical NO detection with a porphyrinic microelectrode to study responses of endothelial cells incubated with homocysteine (Hcy) to the stimulation with bradykinin, calcium ionophore, or L-arginine. Twenty-four-hour preincubation with Hcy (10, 20, and 50 microM) resulted in a gradual decline in responsiveness of endothelial cells to the above stimuli. Hcy did not affect the expression of endothelial nitric oxide synthase (eNOS), but it stimulated formation of superoxide anions, as judged by fluorescence of dichlorofluorescein, and peroxynitrite, as detected by using immunoprecipitation and immunoblotting of proteins modified by tyrosine nitration. Hcy did not directly affect the ability of recombinant eNOS to generate NO, but oxidation of sulfhydryl groups in eNOS reduced its NO-generating activity. Addition of 5-methyltetrahydrofolate restored NO responses to all agonists tested but affected neither the expression of the enzyme nor formation of nitrotyrosine-modified proteins. In addition, a scavenger of peroxynitrite or a cell-permeant superoxide dismutase mimetic reversed the Hcy-induced suppression of NO production by endothelial cells. In conclusion, electrochemical detection of NO release from cultured endothelial cells demonstrated that concentrations of Hcy >20 microM produce a significant indirect suppression of eNOS activity without any discernible effects on its expression. Folates, superoxide ions, and peroxynitrite scavengers restore the NO-generating activity to eNOS, collectively suggesting that cellular redox state plays an important role in HCy-suppressed NO-generating function of this enzyme.

Topics: Animals; Arginine; Bradykinin; Calcimycin; Cells, Cultured; Endothelium, Vascular; Folic Acid; Free Radical Scavengers; Homocysteine; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Rats; Reactive Oxygen Species; Recombinant Proteins; Superoxides; Tyrosine

Vascular aging is mainly characterized by endothelial dysfunction. We found decreased free nitric oxide (NO) levels in aged rat aortas, in conjunction with a sevenfold higher expression and activity of endothelial NO synthase (eNOS). This is shown to be a consequence of age-associated enhanced superoxide (.O(2)(-)) production with concomitant quenching of NO by the formation of peroxynitrite leading to nitrotyrosilation of mitochondrial manganese superoxide dismutase (MnSOD), a molecular footprint of increased peroxynitrite levels, which also increased with age. Thus, vascular aging appears to be initiated by augmented.O(2)(-) release, trapping of vasorelaxant NO, and subsequent peroxynitrite formation, followed by the nitration and inhibition of MnSOD. Increased eNOS expression and activity is a compensatory, but eventually futile, mechanism to counter regulate the loss of NO. The ultrastructural distribution of 3-nitrotyrosyl suggests that mitochondrial dysfunction plays a major role in the vascular aging process.

Topics: Acetylcholine; Aging; Animals; Aorta; Body Weight; Calcimycin; Cellular Senescence; Endothelium, Vascular; Enzyme Induction; Hemodynamics; Male; Microscopy, Immunoelectron; Mitochondria; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitroprusside; Oxidative Stress; Rats; Rats, Inbred Strains; Superoxide Dismutase; Superoxides; Tyrosine; Vasodilation

We measured the amounts of tyrosine and 3-nitrotyrosine (NO2-tyrosine) in proteins of plasma and polymorphonuclear leukocytes (PMN) from human whole blood before and after activation with phorbol ester (PMA) or calcium ionophore (A 23187). In unstimulated blood, no significant nitration of tyrosine was detected into PMN proteins, but a NO2-tyrosine/tyrosine ratio of 0.7% was detected in plasma proteins. When blood was activated with PMA, the NO2-tyrosine/tyrosine ratio stayed at 0.7% in plasma proteins, but it increased to 1.4% in PMN proteins, indicating a peroxynitrite production within the cells. In blood activated with calcium ionophore, the NO2-tyrosine/tyrosine ratio was 1.2% in plasma proteins and 2.1% in PMN proteins. Incubation of blood with a NO-synthase inhibitor before stimulation inhibited such a protein tyrosine nitration. To ensure that NO2-tyrosine detected in intracellular proteins did not result from the enzymatic posttranslational tyrosylation of PMN proteins, the incorporation of 14C labeled tyrosine into PMN proteins after activation with PMA or A23187 was studied. The addition of a 10 fold excess of NO2-tyrosine did not modify the course of protein tyrosylation. Because tyrosine nitration is an irreversible reaction, NO2-tyrosine could be accumulated into proteins and could act as a cumulative index of peroxynitrite production.

Topics: Blood Proteins; Calcimycin; Humans; Ionophores; Kinetics; Neutrophils; Nitrates; Tetradecanoylphorbol Acetate; Time Factors; Tyrosine