Abstract-During the last century, nitroglycerin has been the most commonly used antiischemic and antianginal agent.Unfortunately, after continuous application, its therapeutic efficacy rapidly vanishes. Neurohormonal activation of vasoconstrictor signals and intravascular volume expansion constitute early counter-regulatory responses (pseudotolerance), whereas long-term treatment induces intrinsic vascular changes, eg, a loss of nitrovasodilator-responsiveness (vascular tolerance). This is caused by increased vascular superoxide production and a supersensitivity to vasoconstrictors secondary to a tonic activation of protein kinase C. NADPH oxidase(s) and uncoupled endothelial nitric oxide synthase have been proposed as superoxide sources. Superoxide and vascular NO rapidly form peroxynitrite, which aggravates tolerance by promoting NO synthase uncoupling and inhibition of soluble guanylyl cyclase and prostacyclin synthase. This oxidative stress concept may explain why radical scavengers and substances, which reduce oxidative stress indirectly, are able to relieve tolerance and endothelial dysfunction. Recent work has defined a new tolerance mechanism, ie, an inhibition of mitochondrial aldehyde dehydrogenase, the enzyme that accomplishes bioactivation of nitroglycerin, and has identified mitochondria as an additional source of reactive oxygen species. Nitroglycerin-induced reactive oxygen species inhibit the bioactivation of nitroglycerin by thiol oxidation of aldehyde dehydrogenase. Both mechanisms, increased oxidative stress and impaired bioactivation of nitroglycerin, can be joined to provide a new concept for nitroglycerin tolerance and cross-tolerance. The consequences of these processes for the nitroglycerin downstream targets soluble guanylyl cyclase, cGMP-dependent protein kinase, cGMP-degrading phosphodiesterases, and toxic side effects contributing to endothelial dysfunction, such as inhibition of prostacyclin synthase, are discussed in this review. (Circ Res. 2005;97:618-628.)Key Words: oxidative stress Ⅲ nitroglycerin Ⅲ nitric oxide Ⅲ endothelial dysfunction N itroglycerin (GTN) has been one of the most widely used antiischemic drugs for more than a century. Given acutely, organic nitrates are excellent agents for the treatment of stable-effort angina, unstable angina, in patients with acute myocardial infarction and in patients with chronic congestive heart failure. The chronic efficacy of nitrates, however, is blunted because of the development of nitrate tolerance. 1 The problem of tolerance has been raised since the first clinical reports of nitrate therapy for hypertension in Bright's disease. In 1888, Stewart reported a case of GTN tolerance in a man Mechanisms of GTN Activation and ActionHow Does GTN Induce Vasodilation?There is a major consent that the principle mechanism of GTN-induced smooth muscle relaxation is the activation of the intracellular NO receptor enzyme, soluble guanylyl cyclase (sGC), subsequent elevation of the cyclic GMP (cGMP) levels, and activation of cGMP-dependent prot...

Abstract-Endothelial dysfunction in the setting of cardiovascular risk factors, such as hypercholesterolemia, hypertension, diabetes mellitus, chronic smoking, as well as in the setting of heart failure, has been shown to be at least partly dependent on the production of reactive oxygen species (ROS), such as the superoxide radical, and the subsequent decrease in vascular bioavailability of nitric oxide (NO). Superoxide-producing enzymes involved in increased oxidative stress within vascular tissue include the NAD(P)H oxidase, the xanthine oxidase, and mitochondrial superoxideproducing enzymes. Superoxide produced by the NADPH oxidase may react with NO released by endothelial nitric oxide synthase (eNOS), thereby generating peroxynitrite. Peroxynitrite in turn has been shown to uncouple eNOS, thereby switching an antiatherosclerotic NO-producing enzyme to an enzyme that may initiate or even accelerate the atherosclerotic process by producing superoxide. Increased oxidative stress in the vasculature, however, is not restricted to the endothelium and has also been demonstrated to occur within the smooth muscle cell layer in the setting of hypercholesterolemia, diabetes mellitus, hypertension, congestive heart failure, and nitrate tolerance. Increased superoxide production by the endothelial and/or smooth muscle cells has important consequences with respect to signaling by the soluble guanylyl cyclase (sGC) and the cGMP-dependent protein kinase I (cGK-I), the activity and expression of which has been shown to be regulated in a redox-sensitive fashion. The present review summarizes current concepts concerning eNOS uncoupling and also focuses on the consequences for downstream signaling with respect to activity and expression of the sGC and cGK-I in various diseases. Key Words: endothelium Ⅲ vasodilation Ⅲ nitric oxide Ⅲ endothelial NO-synthase Ⅲ oxidative stress T raditionally, the role of the endothelium was thought primarily to be that of a selective barrier to the diffusion of macromolecules from the vessel lumen to the interstitial space. During the past 20 years, numerous additional roles for the endothelium have been defined such as regulation of vascular tone, modulation of inflammation, promotion, and inhibition of vascular growth and modulation of platelet aggregation and coagulation. Endothelial dysfunction is a characteristic feature of patients with coronary atherosclerosis and more recent studies indicate that it may predict long-term atherosclerotic disease progression as well as cardiovascular event rate. 1 Although the mechanisms underlying endothelial dysfunction may be multifactorial, there is a growing body of evidence that increased production of reactive oxygen species (ROS) may contribute considerably to this phenomenon. ROS production has been demonstrated to occur in the endothelial cell layer, but also within the media and adventitia, all of which may impair nitric oxide (NO) signaling within vascular tissue to endotheliumdependent, but also endothelium-independent, vasodilators. More recent...

We investigated the mechanisms by which cytokines lead to a diminished responsiveness of vascular smooth muscle to vasoconstrictors. The attenuation of noradrenaline‐induced contraction by 6 to 24 h incubations with the cytokines, tumor necrosis factor and interleukin‐1, in endotheliumdenuded rabbi, aorta was associated with an increase in intracellular cyclic GMP level. This increase was abolished by the stereoselective inhibitor of nitric oxide‐synthase. N o‐nitro‐L‐arginine and by cycloheximide. Formation of nitric oxide was detected in the cytosol of cytokine‐treated native and cultured smooth muscle cells by activation of purified soluble guanylate cyclase, and depended on tetrahydrobiopterin, but not on ? ‐calmodulin. The results indicate that cytokines induce a nitric oxide‐synthase of the macrophage‐type in vascular smooth muscle.

We investigated whether calmodulin mediates the stimulating effect of Ca2+ on nitric oxide synthase in the cytosol of porcine aortic endothelial cells. Nitric oxide was quantified by activation of a purified soluble guanylate cyclase. The Ca*+ -sensitivity of nitric oxide synthase was lost after anion exchange chromatography of the endothelial cytosol and could only be. reconstituted by addition of cahnodulin or heat-denatured endothelial cytosol. The CaZf-dependent activation of nitric oxide synthase in the cytosol was inhibited by the calmodulin-binding peptides/proteins melittin, mastoparan, and calcineurin (IC,, 450, 350 and 60 nM, respectively), but not by the calmodulin antagonist, calmidazolium. In contrast, Ca2+-calmodulin-reconstituted nitric oxide synthase was inhibited with similar potency by melittin and calmidazolimn. The results suggest that the CaZ+-dependent activation of nitric oxide synthaae in endothelial cells is mediated by calmodulin.

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D espite impressive medical advances 1 that have led to diminished cardiovascular death rates in some countries over the past 20 years, cardiovascular disease remains the leading cause of death in developed countries such as the United States. This promises to worsen as a result of aging populations; the incipient obesity and type II diabetes epidemic; sedentary lifestyle; and continued abuse of tobacco, alcohol, and other substances. Cardiovascular disease is clearly multifactorial, and the approach to its prevention necessarily likewise. Candidates for prevention include cyclic guanosine 3Ј,5Ј-cyclic monophosphate (cGMP)-dependent signaling networks initiated by natriuretic peptides (NPs) and nitric oxide (NO), which demonstrate characteristics deemed worthy of diagnostic and therapeutic exploitation. cGMP signaling contributes to the function and interaction of several vascular cell types, and its dysfunction could be involved in major destructive processes such as atherosclerosis, hypertension, diabetic complications, (re)stenosis, and tissue infarction, as well as the undermining of clinical therapy like in the case of nitrate tolerance. This review takes a focused look at key elements of the cGMP signaling cascade in vascular tissue, particularly recent advances in our knowledge of cGMP-dependent protein kinase (cGK, also known as PKG) function. Finally, we discuss the potential of clinical monitoring of cGK activity for assessing the functional status of cGMP signaling and for guiding the design of therapeutic strategies to improve vascular function. cGMP SynthesisOne of the 2 major synthetic pathways for cGMP generation from guanosine 5Ј-triphosphate (GTP) is directed by NPs (Figure 1), consisting of atrial (ANP), B-type (BNP), and C-type (CNP) natriuretic peptides, which act via the membrane receptor guanylate cyclases GC-A (highest affinity for ANP, BNP) and GC-B (highest affinity for CNP). ANP and BNP, released from the heart by mechanical stretch in response to increased atrial pressure/volume, and CNP, released from the endothelium, can all cause smooth muscle (SM) relaxation and vasodilation. 2,3 Particularly, ANP has multiple effects, which lead to vascular smooth muscle (VSM) relaxation, increased vascular permeability and glomerular filtration rate, inhibition of the sympathetic and renin-angiotensin-aldosterone systems, and natriuresis/diuresis. 3 Germline deletion of ANP or its GC-A receptor in mice resulted in varying degrees of hypertension, cardiomyopathy, and heart failure (reviewed in reference 2 ), but also cardiac hypertrophy that is independent of hypertension. 4,5 ANP effects on VSM itself were analyzed in transgenic mice engineered to lack GC-A selectively in smooth muscle. These mice displayed resistance to vasodilation by acute ANP treatment or vascular volume expansion, but normal resting blood pressure, 6 suggesting that the function of GC-A in VSM can be compensated for in chronic blood pressure regulation.The second major pathway for cGMP synthesis (Figure 1) entails stimulatio...

The objective of this study was to identify a potential mechanism for S-nitrosation of proteins. Therefore, we assessed S-nitrosation of bovine serum albumin by dinitrosyl-iron-di-L-cysteine complex [(NO) 2 Fe(L-cysteine) 2 ], a compound similar to naturally occurring iron-nitrosyls. Within 5-10 min, (NO) 2 Fe(L-cysteine) 2 generated paramagnetic albumin-bound dinitrosyl-iron complex and S-nitrosoalbumin in a ratio of 4:1. Although S-nitroso-L-cysteine was concomitantly formed in low amounts, its concentration was not sufficient to account for formation of S-nitrosoalbumin via a trans-S-nitrosation reaction. Low oxygen tension did not affect S-nitrosation by the dinitrosyl-iron complex thus excluding the involvement of oxygenated NO x -species in the nitrosation reaction. Blockade of albumin histidine residues by pyrocarbonate, which prevented formation of dinitrosyliron-albumin complex, did not inhibit S-nitrosation of albumin. Thus, S-nitrosation of albumin by (NO) 2 Fe(Lcysteine) 2 can proceed by direct attack of a nitrosyl moiety on the protein thiolate, without previous binding of the iron. We conclude that protein-bound dinitrosyliron complexes detected in high concentrations in certain tissues provide a reservoir of S-nitrosating species, e.g. low molecular dinitrosyl iron complexes.

Mitochondrial aldehyde dehydrogenase (ALDH-2) was recently identified to be essential for the bioactivation of glyceryl trinitrate (GTN). Here we assessed whether other organic nitrates are bioactivated by a similar mechanism. The ALDH-2 inhibitor benomyl reduced the vasodilator potency, but not the efficacy, of GTN, pentaerythritol tetranitrate (PETN), and pentaerythritol trinitrate in phenylephrine-constricted rat aorta, whereas vasodilator responses to isosorbide dinitrate, isosorbide-5-mononitrate, pentaerythritol dinitrate, pentaerythritol mononitrate, and the endothelium-dependent vasodilator acetylcholine were not affected. Likewise, benomyl decreased GTN-and PETN-elicited phosphorylation of the cGMP-activated protein kinase substrate vasodilator-stimulated phosphoprotein (VASP) but not that elicited by other nitrates. The vasodilator potency of organic nitrates correlated with their potency to inhibit ALDH-2 dehydrogenase activity in mitochondria from rat heart and increase mitochondrial superoxide formation, as detected by chemiluminescence. In contrast, mitochondrial ALDH-2 esterase activity was not affected by PETN and its metabolites, whereas it was inhibited by benomyl, GTN applied in vitro and in vivo, and some sulfhydryl oxidants. The bioactivation-related metabolism of GTN to glyceryl-1,2-dinitrate by isolated RAW macrophages was reduced by the ALDH-2 inhibitors benomyl and daidzin, as well as by GTN at concentrations Ͼ1 M. We conclude that mitochondrial ALDH-2, specifically its esterase activity, is required for the bioactivation of the organic nitrates with high vasodilator potency, such as GTN and PETN, but not for the less potent nitrates. It is interesting that ALDH-2 esterase activity was inhibited by GTN only, not by the other nitrates tested. This difference might explain why GTN elicits mitochondrial superoxide formation and nitrate tolerance with the highest potency.Organic nitrates such as nitroglycerin (glyceryl trinitrate; GTN) are widely used in the therapy of cardiovascular diseases such as stable and unstable angina (Abrams, 1995).The anti-ischemic effects of organic nitrates are largely caused by venous and coronary artery dilation as well as the improvement of collateral blood flow, which all decrease myocardial oxygen consumption. Their use, however, is limited because of the rapid development of tolerance and crosstolerance characterized by decreased sensitivity of the vasculature to the organic nitrates and to endothelium-dependent vasodilators, respectively (Mangione and Glasser,