Heme-dependent activation of guanylate cyclase by nitric oxide: a novel signal transduction mechanism.

The interaction between nitric oxide (NO) synthesized in one cell and cytosolic guanylate-cyclase-bound heme located in adjacent target cells to generate the NO-heme adduct of guanylate cyclase represents a novel and widespread signal transduction mechanism that links extracellular stimuli to the biosynthesis of cyclic GMP in target cells. A variety of chemical factors interact with selective extracellular receptors and trigger the biosynthesis of NO from L-arginine. The unique chemistry of NO endows this molecule with the capacity to diffuse rapidly into nearby cells and stimulate cyclic GMP formation. Cyclic GMP acts as a messenger in each cell type to trigger different but complementary cellular responses within a localized environment. This transcellular signaling is a form of rapid intercellular communication allowing the simultaneous local initiation of increased blood flow, inhibition of platelet-induced thrombosis and other cellular functions.     

Formation of free nitric oxide from l-arginine by nitric oxide synthase: direct enhancement of generation by superoxide dismutase.

Although nitric oxide (NO) appears to be one of the oxidation products of L-arginine catalyzed by NO synthase (NOS; EC 1.14.13.39), past studies on the measurement of NO in cell-free enzymatic assays have not been based on the direct detection of the free NO molecule. Instead, assays have relied on indirect measurements of the stable NO oxidation products nitrite and nitrate and on indirect actions of NO such as guanylate cyclase activation and oxyhemoglobin oxidation. Utilizing a specific chemiluminescence assay, we report here that the gaseous product of L-arginine oxidation, catalyzed by both inducible macrophage and constitutive neuronal NOS, is indistinguishable from authentic NO on the basis of their physicochemical properties. NO gas formation by NOS was dependent on L-arginine, NADPH, and oxygen and inhibited by NG-methyl-L-arginine and cyanide anion. Superoxide dismutase (SOD) caused a marked, concentration-dependent increase in the production of free NO by mechanisms that were unrelated to the dismutation of superoxide anion or activation of NOS. These observations indicate that free NO is formed as a result of NOS-catalyzed L-arginine oxidation and that SOD enhances the generation of NO without directly affecting NO itself. SOD appears to elicit a novel biological action, perhaps accelerating the conversion of an intermediate in the L-arginine-NO pathway such as nitroxyl (HNO) to NO.     

Sulfhydryl angiotensin-converting enzyme inhibition induces sustained reduction of systemic oxidative stress and improves the nitric oxide pathway in patients with essential hypertension

Background

Essential hypertension is associated with enhanced LDL oxidation and impaired endothelium-dependent vasodilation. The antioxidant status is linked to the nitric oxide (NO) pathway. Sulfhydryl angiotensin-converting enzyme (ACE) inhibitors inhibit oxidative stress and atherogenesis in experimental models; therefore we tested whether this beneficial antioxidant activity could be also clinically relevant in patients with essential hypertension.

Methods

Plasma LDL oxidizability was investigated initially in untreated normocholesterolemic patients with moderate essential hypertension without clinically evident target organ damage (n = 96) and in control normotensive subjects (n = 46). Patients were then randomly assigned into two age- and sex-matched groups to receive the new sulfhydryl ACE inhibitor zofenopril (15 to 30 mg/d; n = 48) or enalapril (20 mg/d, n = 48). LDL oxidizability was evaluated (generation of malondialdehyde, MDA) and systemic oxidative stress was evaluated by isoprostanes (8-isoPGF_2α). Asymmetrical dimethyl-l-arginine (ADMA), a competitive inhibitor of endothelial NO synthase, and plasma nitrite and nitrates (NOx) were also measured.

Results

LDL from hypertensive subjects had enhanced susceptibility to oxidation in vitro compared with that in control subjects (P < .05). Similarly, isoprostanes were significantly increased (P < .01) in hypertensive subjects versus control subjects. After 12-week treatment, MDA levels were significantly reduced by zofenopril (P < .05) but not enalapril treatment (P = not significant). Isoprostanes were normalized after zofenopril treatment (P < .03), whereas enalapril was ineffective. After treatment with both ACE inhibitors, plasma NOx concentrations were significantly reduced (P < .05). Similarly, hypertension increased ADMA concentration compared with the normotensive state, whereas ACE inihibition elicited a significant decrease. However, the reduction of ADMA concentration was significantly higher in patients receiving sulfhydryl ACE inhibition (P < .05 vs enalapril).

Conclusions

The sulfhydryl ACE inhibitor zofenopril reduces oxidative stress and improves the NO pathway in patients with essential hypertension. If confirmed in a large multicenter clinical trial, our data suggest a possible vasculoprotective effect of the compound in retarding vascular dysfunction and atherogenesis that often develops rapidly in hypertensive patients.     

Oxidation of nitric oxide in aqueous solution to nitrite but not nitrate: comparison with enzymatically formed nitric oxide from L-arginine.

Nitric oxide (NO) in oxygen-containing aqueous solution has a short half-life that is often attributed to a rapid oxidation to both NO2- and NO3-. The chemical fate of NO in aqueous solution is often assumed to be the same as that in air, where NO is oxidized to NO2 followed by dimerization to N2O4. Water then reacts with N2O4 to form both NO2- and NO3-. We report here that NO in aqueous solution containing oxygen is oxidized primarily to NO2- with little or no formation of NO3-. In the presence of oxyhemoglobin or oxymyoglobin, however, NO and NO2- were oxidized completely to NO3-. Methemoglobin was inactive in this regard. The unpurified cytosolic fraction from rat cerebellum, which contains constitutive NO synthase activity, catalyzed the conversion of L-arginine primarily to NO3- (NO2-/NO3- ratio = 0.25). After chromatography on DEAE-Sephacel or affinity chromatography using 2',5'-ADP-Sepharose 4B, active fractions containing NO synthase activity catalyzed the conversion of L-arginine primarily to NO2- (NO2-/NO3- ratio = 5.6) or only to NO2-, respectively. Unpurified cytosol from activated rat alveolar macrophages catalyzed the conversion of L-arginine to NO2- without formation of NO3-. Addition of 30 microM oxyhemoglobin to all enzyme reaction mixtures resulted in the formation primarily of NO3- (NO2-/NO3- ratio = 0.09 to 0.20). Cyanide ion, which displaces NO2- from its binding sites on oxyhemoglobin, inhibited the formation of NO3-, thereby allowing NO2- to accumulate. These observations indicate clearly that the primary decomposition product of NO in aerobic aqueous solution is NO2- and that further oxidation to NO3- requires the presence of additional oxidizing species such as oxyhemoproteins.     

NG-methyl-L-arginine causes endothelium-dependent contraction and inhibition of cyclic GMP formation in artery and vein.

The objective of this study was to determine whether the vascular smooth muscle contractile effect of NG-methyl-L-arginine (NMA) is endothelium dependent and attributed to a decline in smooth muscle levels of cyclic GMP. Vascular smooth muscle levels of cyclic GMP are severalfold greater in endothelium-intact than in endothelium-denuded preparations because of the continuous formation and release of a lipophilic endothelium-derived chemical factor that diffuses into the underlying smooth muscle and activates cytosolic guanylate cyclase. This chemical substance, believed to be nitric oxide (NO) or a labile nitroso precursor, appears to account for the biological actions of endothelium-derived relaxing factor. NMA inhibits the formation of NO from endogenous L-arginine in endothelial cells. In the present study, NMA caused marked endothelium-dependent contraction of isolated rings of bovine pulmonary artery and vein, and this was similar to the contraction elicited by hemoglobin, an inhibitor of the relaxant action of NO. Both NMA and hemoglobin caused endothelium-dependent potentiation of contractile responses to phenylephrine in artery and vein. NMA caused endothelium-dependent decreases in the resting or basal levels of cyclic GMP in artery and vein to levels that were characteristic of those in endothelium-denuded vessels. Finally, NMA inhibited endothelium-dependent relaxant responses and cyclic GMP formation stimulated by acetylcholine and bradykinin. These observations reveal that interference with the continuous or basal generation of endothelium-derived NO in artery and vein can cause marked increases in vascular smooth muscle tone as a result of inhibition of cyclic GMP formation.     

Potential cardioprotective actions of no-releasing aspirin

The use of low doses of aspirin on a daily basis has increased greatly in the past 20 years, based on observations that it can significantly reduce the risk of heart attacks and strokes. However, aspirin can also cause severe damage to the stomach. A modified version of aspirin that releases nitric oxide has been developed that seems to offer important advantages over its 103-year-old parent--namely, improved protection for the heart without the unwanted effects on the stomach.     

Phase II study of pomegranate juice for men with rising prostate-specific antigen following surgery or radiation for prostate cancer.

PURPOSE: Phytochemicals in plants may have cancer preventive benefits through antioxidation and via gene-nutrient interactions. We sought to determine the effects of pomegranate juice (a major source of antioxidants) consumption on prostate-specific antigen (PSA) progression in men with a rising PSA following primary therapy. EXPERIMENTAL DESIGN: A phase II, Simon two-stage clinical trial for men with rising PSA after surgery or radiotherapy was conducted. Eligible patients had a detectable PSA > 0.2 and < 5 ng/mL and Gleason score < or = 7. Patients were treated with 8 ounces of pomegranate juice daily (Wonderful variety, 570 mg total polyphenol gallic acid equivalents) until disease progression. Clinical end points included safety and effect on serum PSA, serum-induced proliferation and apoptosis of LNCaP cells, serum lipid peroxidation, and serum nitric oxide levels. RESULTS: The study was fully accrued after efficacy criteria were met. There were no serious adverse events reported and the treatment was well tolerated. Mean PSA doubling time significantly increased with treatment from a mean of 15 months at baseline to 54 months posttreatment (P < 0.001). In vitro assays comparing pretreatment and posttreatment patient serum on the growth of LNCaP showed a 12% decrease in cell proliferation and a 17% increase in apoptosis (P = 0.0048 and 0.0004, respectively), a 23% increase in serum nitric oxide (P = 0.0085), and significant (P < 0.02) reductions in oxidative state and sensitivity to oxidation of serum lipids after versus before pomegranate juice consumption. CONCLUSIONS: We report the first clinical trial of pomegranate juice in patients with prostate cancer. The statistically significant prolongation of PSA doubling time, coupled with corresponding laboratory effects on prostate cancer in vitro cell proliferation and apoptosis as well as oxidative stress, warrant further testing in a placebo-controlled study.     

Changes in liver lysosome fragility, erythrocyte membrane stability, and local and systemic lysosomal enzyme levels in adjuvant-induced polyarthritis

Adjuvant-induced polyarthritis in rats is characterized by the development of liver lysosome fragility, and elevated plasma and local hind paw activities of lysosomal acid hydrolases. During the first few days of the disease, there is a marked increase in liver lysosome fragility and a sharp elevation in plasma β-glucuronidase and acid phosphatase. Commencing on day 4, both liver lysosome fragility and plasma levels of acid hydrolases decrease. Liver lysosome fragility and plasma acid hydrolases again increase on day 9 and continue to rise. Between days 4 and 9, when liver lysosomes appear to be stabilized and plasma levels of acid hydrolases are near control values, the onset of erythrocyte membrane stabilization occurs which endures throughout the disease. Acid phosphatase activity in the injected hind paw increases immediately after induction of polyarthritis as does the swelling, whereas enzyme activity in and swelling of the contralateral hind paw increase 9 or 10 days later. Paramethasone, phenylbutazone and indomethacin, administered orally for various periods of time, attenuated the swelling of primary and secondary lesions and reduced the severity of the biochemical lesions examined.