Claure N. Lunardi

The antioxidant action of Polypodium leucotomos extract and kojic acid : reactions with reactive oxygen species

Two natural products Polypodium leucotomos extract (PL) and kojic acid (KA) were tested for their ability to scavenge reactive oxygen species (.OH,.O2-, H2O2, 1O2) in phosphate buffer. Hydroxyl radicals were generated by the Fenton reaction, and the rate constants of scavenging were 1.6 x 10(9) M-1 s-1 for KA and 1.0 x 10(9) M-1 s-1 for PL, similar to that of ethanol (1.4 x 10(9) M-1 s-1). With superoxide anions generated by the xanthine/hypoxanthine system, KA and PL (0.2-1.0 mg/ml) inhibited.O2-dependent reduction of nitroblue tetrazolium by up to 30 and 31%, respectively. In the detection of 1O2 by rose bengal irradiation, PL at 1.0 mg/ml quenched singlet oxygen by 43% relative to azide and KA by 36%. The present study demonstrates that PL showed an antioxidant effect, scavenging three of four reactive oxygen species tested here. Unlike KA, PL did not significantly scavenge hydrogen peroxide.

Vitamin C improves the effect of a new nitric oxide donor on the vascular smooth muscle from renal hypertensive rats

Impaired relaxation induced by the new nitric oxide (NO) donor [Ru(NH.NHq)(terpy)NO(+)](3+) (TERPY) has been observed in the aortic rings from renal hypertensive rats (2K-1C). An increased production of reactive oxygen species (ROS) in the aortas from 2K-1C rats are capable of reducing NO bioavailability. Therefore, this study aimed at investigating the effects of an antioxidant (vitamin C) on the relaxant effect of NO released from TERPY on the 2K-1C rat aorta. As for vascular reactivity, the potency of TERPY is greater in the control rats (2K) than in 2K-1C whereas the maximum relaxation (ME) is not significantly different between the 2K and 2K-1C rat aortas. The relaxation of TERPY is potentiated only in the 2K-1C aortic ring treated with vitamin C. TERPY has a lower effect in decreasing cytosolic Ca(2+) concentration ([Ca(2+)]c) in vascular smooth muscle cells (VSMCs) from 2K-1C rats. This effect is also potentiated in 2K-1C aortic cells treated with vitamin C, but it is not altered in 2K cells. The basal cytosolic NO concentration ([NO]c) is lower in 2K-1C than in 2K cells, and the bioavailability of the NO released from TERPY is larger in 2K than in 2K-1C VSMCs. The superoxide radical concentration ([O(2)(*-)]) is higher in the 2K-1C aorta, and vitamin C reduces the [O(2)(*-)] in the 2K-1C aorta. Taken together, these results show that in the aortas of renal hypertensive 2K-1C rats, released NO from the new NO donor is not available to produce a similar effect in 2K aorta due to increased [O(2)(*-)].

New nitric oxide donors based on ruthenium complexes

Nitric oxide (NO) donors produce NO-related activity when applied to biological systems. Among its diverse functions, NO has been implicated in vascular smooth muscle relaxation. Despite the great importance of NO in biological systems, its pharmacological and physiological studies have been limited due to its high reactivity and short half-life. In this review we will focus on our recent investigations of nitrosyl ruthenium complexes as NO-delivery agents and their effects on vascular smooth muscle cell relaxation. The high affinity of ruthenium for NO is a marked feature of its chemistry. The main signaling pathway responsible for the vascular relaxation induced by NO involves the activation of soluble guanylyl-cyclase, with subsequent accumulation of cGMP and activation of cGMP-dependent protein kinase. This in turn can activate several proteins such as K+ channels as well as induce vasodilatation by a decrease in cytosolic Ca2+. Oxidative stress and associated oxidative damage are mediators of vascular damage in several cardiovascular diseases, including hypertension. The increased production of the superoxide anion (O2-) by the vascular wall has been observed in different animal models of hypertension. Vascular relaxation to the endogenous NO-related response or to NO released from NO deliverers is impaired in vessels from renal hypertensive (2K-1C) rats. A growing amount of evidence supports the possibility that increased NO inactivation by excess O2- may account for the decreased NO bioavailability and vascular dysfunction in hypertension.

Caveolae dysfunction contributes to impaired relaxation induced by nitric oxide donor in aorta from renal hypertensive rats.

Relaxation induced by nitric oxide (NO) donors is impaired in renal hypertensive two kidney-one clip (2K-1C) rat aortas. It has been proposed that caveolae are important in signal transduction and Ca2+ homeostasis. Therefore, in the present study we investigate the integrity of caveolae in vascular smooth muscle cells (VSMCs), as well as their influence on the effects produced by NO released from both the new NO donor [Ru(NH.NHq) (terpy)NO+]3+ (TERPY) and sodium nitroprusside (SNP) on 2K-1C rat aorta. The potency of both TERPY and SNP was lower in the 2K-1C aorta that in the normotensive aorta [two kidney (2K)], whereas the maximal relaxant effect (ME) was similar in both 2K-1C and 2K aortas. In the 2K aorta, methyl-β-cyclodextrin (CD) reduced both the potency of TERPY and SNP, and their ME compared with the control, but it had no effect on the potency and ME of these NO donors in 2K-1C aortas. The decrease in cytosolic Ca2+ concentration ([Ca2+]c) induced by TERPY was larger in 2K than in 2K-1C cells, and this effect was inhibited by CD in 2K cells only. Aortic VSMCs from 2K rats presented a larger number of caveolae than those from 2K-1C rats. Treatment with CD reduced the number of caveolae in both 2K and 2K-1C aortic VSMCs. Our results support the idea that caveolae play a critical role in the relaxant effect and in the decrease in [Ca2+]c induced by NO, and they could be responsible for impaired aorta relaxation by NO in renal hypertensive rats.

Nitric oxide release from the S-nitrosothiol zinc phthalocyanine complex by flash photolysis

The photogeneration of nitric oxide (NO) using laser flash photolysis was investigated for S-nitroso-glutathione (GSNO) and S-nitroso-N-acetylcysteine (NacySNO) at pH 6.4 (PBS/HCl) and 7.4 (PBS). Irradiation of S-nitrosothiol with light (lambda = 355 nm followed by absorption spectroscopy) resulted in the homolytic decomposition of NacySNO and GSNO to generate radicals (GS and NacyS ) and NO. The release of NO from donor compounds measured with an ISO-Nometer apparatus was larger at pH 7.4 than pH 6.4. NacySNO was also incorporated into dipalmitoyl-phosphatidylcholine liposomes in the presence and absence of zinc phthalocyanine (ZnPC), a well-known photosensitizer useful for photodynamic therapy. Liposomes are usually used as carriers for hydrophobic compounds such as ZnPC. Inclusion of ZnPC resulted in a decrease in NO liberation in liposomal medium. However, there was a synergistic action of both photosensitizers and S-nitrosothiols resulting in the formation of other reactive species such as peroxynitrite, which is a potent oxidizing agent. These data show that NO release depends on pH and the medium, as well as on the laser energy applied to the system. Changes in the absorption spectrum were monitored as a function of light exposure.

The inducing NO-vasodilation by chemical reduction of coordinated nitrite ion in cis-[Ru(NO2)L(bpy)2]+ complex

The synthesis of [Ru(NO(2))L(bpy)(2)](+) (bpy = 2,2-bipyridine and L = pyridine (py) and pyrazine (pz)) can be accomplished by addition of [Ru(NO)L(bpy)(2)](PF(6))(3) to aqueous solutions of physiological pH. The electrochemical processes of [Ru(NO(2))L(bpy)(2)](+) in aqueous solution were studied by cyclic voltammetry and differential pulse voltammetry. The anodic scan shows a peak around 1.00 V vs. Ag/AgCl attributed to the oxidation process centered on the metal ion. However, in the cathodic scan a second peak around -0.60 V vs. Ag/AgCl was observed and attributed to the reduction process centered on the nitrite ligand. The controlled reduction potential electrolysis at -0.80 V vs. Ag/AgCl shows NO release characteristics as judged by NO measurement with a NO-sensor. This assumption was confirmed by ESI/MS(+) and spectroelectrochemical experiment where cis-[Ru(bpy)(2)L(H(2)O)](2+) was obtained as a product of the reduction of cis-[Ru(II)(NO(2))L(bpy)(2)](+). The vasorelaxation observed in denuded aortic rings pre-contracted with 0.1 mumol L(-1) phenylephrine responded with relaxation in the presence of cis-[Ru(II)(NO(2))L(bpy)(2)](+). The potential of rat aorta cells to metabolize cis-[Ru(II)(NO(2))L(bpy)(2)](+) was also followed by confocal analysis. The obtained results suggest that NO release happens by reduction of cis-[Ru(II)(NO(2))L(bpy)(2)](+) inside the cell. The maximum vasorelaxation was achieved with 1 x 10(-5) mol L(-1) of cis-[Ru(II)(NO(2))L(bpy)(2)](+) complex.

Relaxation induced by calcium ionophore is impaired in carotid arteries from 2K-1C rats due to failed effect of nitric oxide on the smooth muscle cells.

Vascular endothelium generates nitric oxide (NO) in large vessels and induces relaxation of vascular smooth muscle cells (VSMC). The aim of this study was to evaluate the contribution of NO produced in the endothelial cells (EC) to the relaxation induced by the Ca2+ ionophore A23187 and whether this relaxation is impaired in renal hypertensive (2K-1C) rat arteries. Concentration-effect curves for A23187 were constructed in intact endothelium isolated carotid rings from 2K-1C and normotensive (2K) in the absence or in the presence of the extracellular NO scavenger haemoglobin or inhibitors of NO-synthase (NOS, L-NOARG), guanylyl-cyclase (GC, ODQ). In carotid rings loaded with Fluo-3AM, both EC and VSMC were simultaneously imaged by a confocal microscope and [Ca2+]c was derived from fluorescence intensities (IF). The maximal relaxation (ME) induced by A23187 was lower in 2K-1C than in 2K arteries. A23187-induced relaxation was abolished by haemoglobin and L-NOARG in both groups. ODQ reduced the ME to A23187 in 2K and abolished its relaxation in 2K-1C. A23187 increased [Ca2+]c in a similar way in 2K and 2K-1C EC, and decreased [Ca2+]c in VSMC, which effect was higher in 2K than in 2K-1C arteries. L-NOARG inhibited the effect of A23187 in VSMC from 2K and abolished it in 2K-1C rats. On the other hand, L-NOARG did not modify the effect of A23187 in EC from 2K and 2K-1C rats. The basal content of cGMP was higher in 2K than in 2K-1C arterial rings that was similarly increased by A23187. In conclusion, the Ca2+ ionophore A23187 increases Ca2+, activates NOS and NO production in the EC activating GC in VSMC and [Ca2+]c decrease. All these effects are higher in 2K, which contribute to the impaired relaxation to A23187 in 2K-1C rat arteries.

Zinc tetranitrophthalocyanine: isomer separation and photophysical photobiological evaluation in J774A tumor cells

The synthesis of zinc tetranitrophthalocyanine (T4NO2ZnPc) was carried out and two structural isomers (C2v and Cs) were separated by column chromatography and characterized by 400 MHz proton nuclear resonance spectroscopy and absorption spectroscopy. The photophysical and photobiological parameters for isomers C2v and Cs were determined in solution and in the presence of macrophage cell culture (J774A). The results obtained clearly indicated that the photophysical, photochemical and photobiological properties of the two isomers were different and each isomer showed a different biological behavior.

Immunoreactivity for neuronal NOS and fluorescent indication of NO formation in the NTS of juvenile rats submitted to chronic intermittent hypoxia.

Exposure to chronic intermittent hypoxia (CIH) leads to significant autonomic and respiratory changes, similar to those observed in obstructive sleep apnea. The hypertension associated with CIH is due to sympathoexcitation triggered by long-term exposure to intermittent hypoxia. However, the mechanisms underlying these effects are unknown. Changes in central regulation of sympathetic activity may underlie CIH-induced hypertension. Since NO appears to be mainly sympathoinhibitory in the nucleus of the solitary tract (NTS), we hypothesized that CIH augments sympathetic activity, in part by reducing neuronal nitric oxide synthase (nNOS) expression and consequently nitric oxide (NO) production in this brain region. To test our hypothesis, juvenile male Wistar rats were exposed to CIH for 8 h/day for 10 days and sections of perfused brainstem were either stained to reveal nNOS-immunoreactivity or loaded with DAF 2-DA to label neurons containing NO. CIH rats showed a significant increase in mean arterial pressure and heart rate compared to controls. However, there was no significant difference in the distribution, staining intensity or numbers of nNOS-immunoreactive neurons in the NTS between experimental and control rats. We also found no significant change in NO content in the DAF 2-DA-loaded sections of NTS from CIH rats. Our data show that NO is not altered in the NTS of juvenile CIH rats, suggesting that nitrergic mechanisms, at least in the NTS, are unlikely to be involved in the sympathetic excitation that generates the hypertension observed after 10 days of CIH.

The complex between 9-(n-decanyl)acridone and Bovine Serum Albumin. Part 2. What do fluorescence probes probe?For Part 1 see ref. 1.

Factor analysis indicates that the fluorescence spectrum of 9-(n-decanyl)acridone (NDA), when bound to Bovine Serum Albumin (BSA), can be described quite adequately as the sum of two spectra, attributed to a free and a bound species. Kinetic evidence indicates that upon electronic excitation the system undergoes a net increase in free NDA, relative to the equilibrium distribution in the ground state, which would be consistent with Lewis acid sites on BSA being responsible for the binding. The system does not attain a position of equilibrium during the duration of the excited singlet state. This permits the determination of excited state rate constants for binding and unbinding of NDA on BSA, as well as the decay constants for the two forms of the probe.