Nitric oxide is an important bioregulatory molecule, being responsible, for example, for activity of endothelium-derived relaxing factor (EDRF). Acute hypertension, diabetes, ischaemia and atherosclerosis are associated with abnormalities of EDRF. Nitric oxide is thought to be a retrograde messenger in the central nervous system. The technology is not yet available for rapid detection of NO released by a single cell in the presence of oxygen and/or nitrite, so the release, distribution and reactivity of endogenous NO in biological systems cannot be analysed. Here we describe a porphyrinic microsensor that we have developed and applied to monitoring NO release in a microsystem. We selectively measured in situ the NO released from a single cell with a response time of less than 10 ms. The microsensor consists of p-type semiconducting polymeric porphyrin and a cationic exchanger (Nafion) deposited on a thermally sharpened carbon fibre with a tip diameter of approximately 0.5 microns. The microsensor, which can be operated in either the amperometric or voltammetric mode, is characterized by a linear response up to 300 microM and a detection limit of 10 nM. Nitric oxide at the level of 10(-20) mols can be detected in a single cell.

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 (·O2 −) 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 ·O2 − 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.

1 Ever increasing use of engineered carbon nanoparticles in nanopharmacology for selective imaging, sensor or drug delivery systems has increased the potential for blood platelet-nanoparticle interactions.2 We studied the effects of engineered and combustion-derived carbon nanoparticles on human platelet aggregation in vitro and rat vascular thrombosis in vivo. 3 Multiplewall (MWNT), singlewall (SWNT) nanotubes, C60 fullerenes (C60CS) and mixed carbon nanoparticles (MCN) (0.2-300 mg ml À1 ) were investigated. Nanoparticles were compared with standard urban particulate matter (SRM1648, average size 1.4 mm). 4 Platelet function was studied using lumi aggregometry, phase-contrast, immunofluorescence and transmission electron microscopy, flow cytometry, zymography and pharmacological inhibitors of platelet aggregation. Vascular thrombosis was induced by ferric chloride and the rate of thrombosis was measured, in the presence of carbon particles, with an ultrasonic flow probe. 5 Carbon particles, except C60CS, stimulated platelet aggregation (MCNXSWNT4MWNT4 SRM1648) and accelerated the rate of vascular thrombosis in rat carotid arteries with a similar rank order of efficacy. All particles resulted in upregulation of GPIIb/IIIa in platelets. In contrast, particles differentially affected the release of platelet granules, as well as the activity of thromboxane-, ADP, matrix metalloproteinase-and protein kinase C-dependent pathways of aggregation. Furthermore, particle-induced aggregation was inhibited by prostacyclin and S-nitroso-glutathione, but not by aspirin. 6 Thus, some carbon nanoparticles and microparticles have the ability to activate platelets and enhance vascular thrombosis. These observations are of importance for the pharmacological use of carbon nanoparticles and pathology of urban particulate matter.

Constitutive nitric oxide synthase (cNOS) with insufficient cofactor (6R)-5,6,7,8-tetrahydrobiopterin (H4B) may generate damaging superoxide (O2-). This study was designed to determine whether cNOS-dependent generation of O2- occurs in spontaneously hypertensive rats (SHR) before the onset of hypertension. Aortas from 4-wk-old SHR and Wistar-Kyoto rats were used. cNOS was stimulated by calcium ionophore A23187. In situ measurements of nitric oxide and hydrogen peroxide by electrochemical sensors and O2- production by chemiluminescence method were performed. Isometric tension was continuously recorded. H4B by high performance liquid chromatography and [3H]citrulline assay were determined in homogenized tissue. The A23187-stimulated production of O2- and its superoxide dismutase product hydrogen peroxide were significantly higher, whereas nitric oxide release was reduced in SHR aortas, with opposite results in the presence of exogenous H4B. Furthermore, NG-monomethyl-L-arginine inhibited the generation of cNOS-dependent O2- by approximately 70%. Natural H4B levels were similar in both strains; however, equivalent cNOS activity required additional H4B in SHR. The endothelium-dependent relaxations to A23187 were significantly inhibited by catalase, and enhanced by superoxide dismutase, only in SHR; however, these enzymes had no effect in the presence of H4B. Thus, dysfunctional cNOS may be a source of O2- in prehypertensive SHR and contribute to the development of hypertension and its vascular complications.

Background-NO regulates vascular tone and structure, platelets, and monocytes. NO is synthesized by endothelial NO synthase (eNOS). Endothelial dysfunction occurs in atherosclerosis. Methods and Results-With a porphyrinic microsensor, NO release was measured in atherosclerotic human carotid arteries and normal mammary arteries obtained during surgery. eNOS protein expression was analyzed by immunohistochemistry. In normal arteries, the initial rate of NO release after stimulation with calcium ionophore A23187 (10 mol/L) was 0.42Ϯ0.05 (mol/L)/s (nϭ10). In contrast, the initial rate of NO release was markedly reduced in atherosclerotic segments, to 0.08Ϯ0.04 (mol/L)/s (nϭ10, PϽ0.0001). NO peak concentration in normal arteries was 0.9Ϯ0.09 mol/L (nϭ10) and in atherosclerotic segments, 0.1Ϯ0.03 mol/L (nϭ10, PϽ0.0001). Reduced NO release in atherosclerotic segments was accompanied by marked reduction of immunoreactive eNOS in luminal endothelial cells, although specific endothelial cell markers (CD31) were present (nϭ13). Endothelial cells of vasa vasorum of atherosclerotic segments, however, remained positive for eNOS, as was the endothelium of normal arteries. Conclusions-In clinically relevant human atherosclerosis, eNOS protein expression and NO release are markedly reduced. This may be involved in the progression of atherosclerosis. (Circulation. 1998;97:2494-2498.)

L-Arginine treatment decreased superoxide generation by cNOS while increasing NO accumulation, leading to protection from constriction (microvessel area, 17.77+/-0.95 versus 11.66+/-2.21 microm2 untreated, P<.0005) and reduction of edema after reperfusion (interfiber area, 16.56+/-2.13% versus 27.68+/-7.70% untreated, P<.005).

Abstract-Recently, we demonstrated that the heptapeptide angiotensin-(1-7) (Ang- [1][2][3][4][5][6][7]) exhibits a favorable kinetic of nitric oxide (NO) release accompanied by extremely low superoxide (O 2 Ϫ ) production. In this report we describe AVE 0991, a novel nonpeptide compound that evoked effects similar to Ang-(1-7) on the endothelium. AVE 0991 and unlabeled Ang-(1-7) competed for high-affinity binding of [125 I]-Ang-(1-7) to bovine aortic endothelial cell membranes with IC 50 values of 21Ϯ35 and 220Ϯ280 nmol/L, respectively. Stimulated NO and O 2 Ϫ release from bovine aortic endothelial cells was directly and simultaneously measured on the cell surface by selective electrochemical nanosensors. Peak concentrations of NO and O 2 Ϫ release by AVE 0991 and Ang-(1-7) (both 10 mol/L) were not significantly different (NO: 295Ϯ20 and 270Ϯ25 nmol/L; O 2 Ϫ : 18Ϯ2 and 20Ϯ4 nmol/L). However, the released amount of bioactive NO was Ϸ5 times higher for AVE 0991 in comparison to Ang-(1-7). The selective Ang-(1-7) antagonist [D-Ala 7 ]-Ang-(1-7) inhibited the AVE 0991-induced NO and O 2 Ϫ production by Ϸ50%. A similar inhibition level was observed for the Ang II AT 1 receptor antagonist EXP 3174. In contrast, the Ang II AT 2 receptor antagonist PD 123,177 inhibited the AVE 0991-stimulated NO production by Ϸ90% but without any inhibitory effect on O 2 Ϫ production. Both NO and O 2 Ϫ production were inhibited by NO synthase inhibition (Ϸ70%) and by bradykinin B 2 receptor blockade (Ϸ80%). AVE 0991 efficiently mimics the effects of Ang-(1-7) on the endothelium, most probably through stimulation of a specific, endothelial Ang-(1-7)-sensitive binding site causing kinin-mediated activation of endothelial NO synthase.

Abstract-The stimulation of endothelium-dependent NO release by angiotensin-(1-7) ] has been indirectly shown in terms of vasodilation, which was diminished by NO synthase inhibition or removal of the endothelium. However, direct measurement of endothelium-derived NO has not been analyzed. With a selective porphyrinic microsensor, NO release was directly assessed from single primary cultured bovine aortic endothelial cells. Ang-(1-7) caused a concentration-dependent release of NO of 1 to 10 mol/L, which was attenuated by NO synthase inhibition.[D-Ala 7 ]Ang-(1-7) (5 mol/L), described as a selective antagonist of Ang-(1-7) receptors, inhibited Ang-(1-7)-induced NO release only by Ϸ50%, whereas preincubation of bovine aortic endothelial cells with the angiotensin II subtype 1 and 2 receptor antagonists EXP 3174 and PD 123,177 (both at 0.1 mol/L) led to an inhibition of 60% and 90%, respectively. A complete blockade of the Ang-(1-7)-induced NO release was observed on preincubation of the cells with 1 mol/L concentration of the bradykinin subtype 2 receptor antagonist icatibant (HOE 140), suggesting an important role of local kinins in the action of Ang-(1-7). Simultaneous direct measurement of superoxide (O 2 Ϫ ) detected by an O 2 Ϫ -sensitive microsensor revealed that the moderately Ang-(1-7)-stimulated NO release was accompanied by a very slow concomitant O 2 Ϫ production with a relative low peak concentration in comparison to the O 2 Ϫ production of the strong NO releasers bradykinin and, especially, calcium ionophore. Thus, Ang-(1-7) might preserve the vascular system, among others, due to its low formation of cytotoxic peroxynitrite by the reaction between NO and O 2 Ϫ . (Hypertension. 2001;37:72-76.)