Michael Preik

Plasma nitrite rather than nitrate reflects regional endothelial nitric oxide synthase activity but lacks intrinsic vasodilator action

The plasma level of NOx, i.e., the sum of NO2− and NO3−, is frequently used to assess NO bioavailability in vivo. However, little is known about the kinetics of NO conversion to these metabolites under physiological conditions. Moreover, plasma nitrite recently has been proposed to represent a delivery source for intravascular NO. We therefore sought to investigate in humans whether changes in NOx concentration are a reliable marker for endothelial NO production and whether physiological concentrations of nitrite are vasoactive. NO2− and NO3− concentrations were measured in blood sampled from the antecubital vein and brachial artery of 24 healthy volunteers. No significant arterial-venous gradient was observed for either NO2− or NO3−. Endothelial NO synthase (eNOS) stimulation with acetylcholine (1–10 μg/min) dose-dependently augmented venous NO2− levels by maximally 71%. This effect was paralleled by an almost 4-fold increase in forearm blood flow (FBF), whereas an equieffective dose of papaverine produced no change in venous NO2−. Intraarterial infusion of NO2− had no effect on FBF. NOS inhibition (NG-monomethyl-l-arginine; 4–12 μmol/min) dose-dependently reduced basal NO2− and FBF and blunted acetylcholine-induced vasodilation and NO release by more than 80% and 90%, respectively. In contrast, venous NO3− and total NOx remained unchanged as did systemic arterial NO2− and NO3− levels during all these interventions. FBF and NO release showed a positive association (r = 0.85; P < 0.001). These results contradict the current paradigm that plasma NO3− and/or total NOx are generally useful markers of endogenous NO production and demonstrate that only NO2− reflects acute changes in regional eNOS activity. Our results further demonstrate that physiological levels of nitrite are vasodilator-inactive.

Evidence for in vivo transport of bioactive nitric oxide in human plasma

Although hitherto considered as a strictly locally acting vasodilator, results from recent clinical studies with inhaled nitric oxide (NO) indicate that NO can exert effects beyond the pulmonary circulation. We therefore sought to investigate potential remote vascular effects of intra-arterially applied aqueous NO solution and to identify the mechanisms involved. On bolus application of NO into the brachial artery of 32 healthy volunteers, both diameter of the downstream radial artery and forearm blood flow increased in a dose-dependent manner. Maximum dilator responses were comparable to those after stimulation of endogenous NO formation with acetylcholine and bradykinin. Response kinetics and pattern of NO decomposition suggested that despite the presence of hemoglobin-containing erythrocytes, a significant portion of NO was transported in its unbound form. Infusion of NO (36 micromol/min) into the brachial artery increased levels of plasma nitroso species, nitrite, and nitrate in the draining antecubital vein (by < 2-fold, 30-fold, and 4-fold, respectively), indicative of oxidative and nitrosative chemistry. Infused N-oxides were inactive as vasodilators whereas S-nitrosoglutathione dilated conduit and resistance arteries. Our results suggest that NO can be transported in bioactive form for significant distances along the vascular bed. Both free NO and plasma nitroso species contribute to the dilation of the downstream vasculature.

Plasma Nitrosothiols Contribute to the Systemic Vasodilator Effects of Intravenously Applied NO Experimental and Clinical Study on the Fate of NO in Human Blood

Abstract— Higher doses of inhaled NO exert effects beyond the pulmonary circulation. How such extrapulmonary effects can be reconciled with the presumed short half-life of NO in the blood is unclear. Whereas erythrocytes have been suggested to participate in NO transport, the exact role of plasma in NO delivery in humans is not clear. Therefore, we investigated potential routes of NO decomposition and transport in human plasma. NO consumption in plasma was accompanied by a concentration-dependent increase in nitrite and S-nitrosothiols (RSNOs), with no apparent saturation limit up to 200 &mgr;mol/L. The presence of red blood cells reduced the formation of plasma RSNOs. Intravenous infusion of 30 &mgr;mol/min NO in healthy volunteers increased plasma levels of RSNOs and induced systemic hemodynamic effects at the level of both conduit and resistance vessels, as reflected by dilator responses in the brachial artery and forearm microvasculature. Intravenous application of S-nitrosoglutathione, a potential carrier of bioactive NO, mimicked the vascular effects of NO, whereas nitrite and nitrate were inactive. Changes in plasma nitrosothiols were correlated with vasodilator effects after intravenous application of S-nitrosoglutathione and NO. These findings demonstrate that in humans the pharmacological delivery of NO solutions results in the transport and delivery of NO as RSNOs along the vascular tree.

Serum nitrite sensitively reflects endothelial NO formation in human forearm vasculature: evidence for biochemical assessment of the endothelial l-arginine–NO pathway

OBJECTIVE A reduced bioactivity of endothelial nitric oxide (NO) has been implicated in the pathogenesis of atherosclerosis. In humans, the endothelial L-arginine-NO pathway has been indirectly assessed via the flow response to endothelium-dependent vasodilators locally administered into the coronary, pulmonary or forearm circulation. However, biochemical quantification of endothelial NO formation in these organ circulations has been hampered so far because of the rapid metabolism of NO. Therefore, we aimed to work out a reliable biochemical index to assess endothelial NO formation in human circulation. METHODS In 33 healthy volunteers, forearm blood flow (FBF) was measured by standard techniques of venous occlusion plethysmography at rest, after local application of the endothelium-dependent vasodilator acetylcholine (ACH), the endothelium-independent vasodilator papaverine (PAP), the stereospecific inhibitor of endothelial NO synthase (eNOS) L-NMMA, and L-arginine (ARG), the natural substrate of eNOS. In parallel, nitrite and nitrate concentrations in blood samples taken from the antecubital vein were measured by HPLC using anion-exchange chromatography in combination with electrochemical and ultraviolet detection following a specific sample preparation method. RESULTS ACH dose-dependently increased resting FBF (from 3.0 +/- 0.3 to 10.4 +/- 0.9 ml/min per 100 ml tissue) and serum nitrite concentration (from 402 +/- 59 to 977 +/- 82 nmol/l, both p < 0.05, n = 12). A significant correlation was observed between the changes in FBF and the serum nitrite concentration (r = 0.61, p < 0.0001). L-NMMA reduced resting FBF and endothelium-dependent vasodilation by 30% and this was paralleled by a significant reduction in serum nitrite concentration at the highest dose of ACH (n = 9, p < 0.001). PAP increased FBF more than fourfold, but did not affect serum nitrite concentration (n = 11), whereas ARG significantly increased both FBF and nitrite. Basal serum nitrate amounted to 25 +/- 4 mumol/l and remained constant during the application of ACH, PAP and L-NMMA. CONCLUSIONS The concentration of serum nitrite sensitively reflects changes in endothelial NO formation in human forearm circulation. This biochemical measure may help to characterize the L-arginine-NO pathway in disease states associated with endothelial dysfunction and to further elucidate its pathophysiological significance for the development of atherosclerosis in humans.

Evidence for a Multifactorial Process Involved in the Impaired Flow Response to Nitric Oxide in Hypertensive Patients With Endothelial Dysfunction

The assessment of endothelial function in hypertensive patients receiving acetylcholine has revealed conflicting results. Whether an impaired flow response to acetylcholine is explained solely by a diminished endothelial synthesis of nitric oxide (NO) remains unclear as yet. In the present study, we tested the hypothesis that mechanisms other than reduced NO synthesis contribute to the hypertension-associated impairment of endothelium-dependent vasodilation. Therefore, the dilatory response to endogenous and exogenous NO was measured in resistance arteries and cutaneous microvessels in the forearm circulation of 12 normotensive individuals and 17 hypertensive patients. In addition, the overall dilatory capacity was assessed by peak flow during reactive hyperemia after 3 minutes of ischemia. Forearm blood flow was quantified by venous occlusion plethysmography at rest, during application of the NO donor sodium nitroprusside, and during stimulation of endogenous NO synthesis by acetylcholine and bradykinin. Blood flow velocity in the cutaneous microvasculature was measured with laser-Doppler flowmetry in parallel. Resting forearm flow was comparable in both groups (3.1 +/- 0.2 and 3.4 +/- 0.2 mL.min-1.100mL-1 tissue), whereas blood pressure and thus peripheral vascular resistance was significantly elevated in hypertensive compared with normotensive subjects. Hyperemic peak flow was significantly blunted in hypertensive patients. Sodium nitroprusside, acetylcholine, and bradykinin increased flow in a dose-dependent manner to a comparable extent in the control group (13.3 +/- 0.8, 13.6 +/- 1.3, and 14.6 +/- 0.7 mL.min-1.100mL-1 tissue, respectively). In contrast, in hypertensive patients maximum increase in resting flow was significantly reduced (sodium nitroprusside, -36%; acetylcholine, -44%; and bradykinin, -56%). The flow response after stimulation of endogenous NO synthesis by bradykinin was significantly more blunted compared with that of exogenous NO after application of sodium nitroprusside. In the cutaneous microvasculature, bradykinin-induced increases in blood flow velocity were selectively impaired in hypertensive patients, whereas flow response to acetylcholine was preserved. Thus, we conclude that in arterial hypertension endothelium-dependent, NO-mediated dilation of resistance arteries and cutaneous microvessels of the forearm vasculature is heterogeneously impaired, depending on the type of endothelial receptor stimulated. Furthermore, the present data suggest that in hypertensive patients the impairment of NO-dependent dilation of resistance arteries is caused by at least three different mechanisms: (1) a reduced endothelial synthesis of NO due to either a disturbed signal-transduction pathway and/or a reduced activity of NO synthase, (2) an accelerated NO degradation within the vessel wall, and (3) alterations in the vessel architecture resulting in an overall reduced dilatory capacity of resistance arteries.

Impaired effectiveness of nitric oxide-donors in resistance arteries of patients with arterial hypertension

Objective To assess the dilatory effectiveness of nitric oxide donors in resistance arteries of patients with arterial hypertension in comparison with that in those of normotensive controls Background Endothelium-dependent vasodilation has been demonstrated to be impaired in arterial hypertension. Besides disturbances in endothelial nitric oxide production a reduced vasodilatory effectiveness of nitric oxide might contribute to this phenomenon of endothelial dysfunction. We therefore investigated the dilatory responsiveness of resistance arteries to exogenous nitric oxide by means of administration of the nitric oxide donors glycerol trinitrate (GTN), isosorbide dinitrate (ISDN) and sodium nitroprusside (SNP) in hypertensive patients Methods Forearm blood flow was measured by venous occlusion plethysmography at rest and during intra-arterial infusion of nitric oxide donors at increasing doses in 11 patients with arterial hypertension and in 10 age-matched normotensive controls Results Forearm blood flow at rest was comparable in the two groups and was dose-dependently increased by administration of either nitric oxide donor. In patients with arterial hypertension, blood flow responses to infusions of organic nitrates were significantly impaired over the entire dose-response curve compared with those of normotensive controls (220nmol/min GTN 13.1 ± 1.3 and 8.6 ± 0.3 ml/min per 100 ml tissue; 212nmol/min ISDN 9.9 ± 0.7 and 5.8 ± 1.0 ml/min per 100 ml tissue). Blood flow responses to infusion of the nitric oxide donor SNP were also profoundly impaired in the hypertensive patients, the extent of which impairment equalled that found with the organic nitrates. Within the entire set of normotensive and hypertensive subjects, maximal flow responses to either nitric oxide donor were inversely correlated with mean arterial blood pressure Conclusions Dilation of resistance arteries in response to infusion of nitric oxide donors is impaired in hypertensive patients and the degree of this impairment depends critically on the severity of arterial hypertension. The reduced effectiveness of nitric oxide appears to be independent of the class of nitric oxide donor and thus of the mode of intravascular nitric oxide generation. These findings are likely to have important implications not only for our understanding of the pathophysiological mechanisms of endothelial dysfunction but also for nitric oxide donor therapy in arterial hypertension

Reduction of peripheral flow reserve impairs endothelial function in conduit arteries of patients with essential hypertension

Background A diminished flow reserve in resistance vessels is a hallmark of hypertensive microvascular disease. Hypertension is associated with structural alterations in the microcirculation and a reduced endothelium-dependent dilation in conduit arteries. Both have been demonstrated to predict future cardiovascular events. Objective We hypothesized that a reduced peripheral flow reserve impairs endothelial function in upstream conduit arteries in patients with arterial hypertension. Design In 43 hypertensive patients (HT) and 38 normotensive controls (NT) endothelial function of the brachial artery was assessed by measurement of flow-mediated dilatation (FMD), using high-resolution ultrasound. Peripheral flow reserve (FR) was determined via measurements of forearm blood flow at rest and during increments of reactive hyperaemia, using venous occlusion plethysmography. Results FMD was markedly impaired in HT (3.6 ± 0.3%) as compared with NT (10.2 ± 0.3%), whereas maximum brachial artery diameter following endothelium-independent dilatation was similar in both groups. In hypertensive patients FR was significantly reduced (HT, 3.2 versus NT, 6.0) during reactive hyperaemia after 5 min of ischaemia. FR was associated with FMD (r = 0.68, P < 0.01). Multiple stepwise regression analysis identified FR as a strong independent variable determining the extent of FMD (r2 = 0.46, P < 0.01). In HT the dose–response curve of FMD upon stepwise increases of FR was shifted significantly to the right. Normalization of FR improved FMD in HT by more than 60%. Conclusions In essential hypertension a reduced FR contributes to the endothelial dysfunction of upstream conduit arteries. These findings may have therapeutic and prognostic implications in patients with arterial hypertension.

Direct biochemical evidence for eNOS stimulation by bradykinin in the human forearm vasculature

Abstract.Objective: Although it has been shown recently that acetylcholine (ACh)-induced vasodilation of forearm resistance vessels is predominantly mediated by nitric oxide, direct biochemical evidence for eNOS stimulation by bradykinin (BK) in the human arterial circulation is still lacking. Therefore, the present study was designed to test the hypothesis that in the human forearm vasculature eNOS stimulation significantly contributes to BK-induced vasodilation. Methods: BK was infused in the presence and absence of the NOS inhibitor L-NMMA (8 μmol/min) into the brachial artery of 16 healthy volunteers and the effects compared to muscarinergic eNOS stimulation following acetylcholine infusion. Forearm blood flow (FBF) was measured by venous occlusion plethysmography, and plasma nitrite (NO2−), which represents a sensitive and specific marker of regional eNOS activity, was determined in the antecubital vein and brachial artery by flow injection analysis. Nitric oxide production was calculated as product of the veno-arterial difference of NO2− concentration times FBF. Results: Kininergic (BK: 20, 60, 200 ng/min) as well as muscarinergic (ACh: 1, 3, 10 μg/min) stimulation resulted in a dose-dependent increase in FBF and NO2− in each individual. The relationship between FBF and NO production upon BK infusion was comparable to that obtained with ACh (r = 0.98; n = 64, p < 0.01). Moreover, NOS inhibition reduced both flow responses and NO production (BK: 54 and 75 %; ACh: 57 and 72 %) to a similar extent. Conclusions: These data provide direct biochemical evidence for the involvement of eNOS in bradykinin-induced vasodilation of forearm resistance vessels in humans.

Additive Effect of Coexistent Type 2 Diabetes and Arterial Hypertension on Endothelial Dysfunction in Resistance Arteries of Human Forearm Vasculature

Population studies suggest that vascular complications accumulate when arterial hyper tension supervenes on diabetes mellitus. Although it has been demonstrated that endothelial function is impaired in patients with either diabetes mellitus or arterial hypertension it is unknown whether or not both diseases exert additive effects on endothelial dysfunction. The authors therefore investigated endothelium-dependent and endothelium-independent vasodilation in the forearm vasculature of 44 individuals: in 10 type 2 diabetic patients (DM), in 12 patients with arterial hypertension (HT), in 10 patients with both DM and HT (DM+HT), and in 12 healthy control subjects (C). Forearm blood flow (FBF) was measured by venous occlusion plethysmography at rest and following intraarterial infusion of acetylcholine (ACh) and the NO-donor sodium nitroprusside (SNP) at increasing doses. FBF at rest was significantly lower in diabetic patients: 2.2 ±0.1 (DM) and 2.6 ±0.2 (DM+HT) versus 3.1 ±0.1 (HT) and 3.4 ±0.2 (C) mL/min per 100 mL of tissue. ACh and SNP both increased FBF dose-dependently in each group. The maximum response to ACh was progressively decreased in DM and HT: 13.7 (C) > 8.1 (DM) > 7.6 (HT) > and 5.7 (DM+HT) mL/min per 100 mL of (continued on next page) tissue. Reduction of the endothelium-dependent flow reserve assessed as percent increase in maximum FBF was also impaired following the same rank order: 349 (C) > 268 (DM) > 160 (HT) > 126 (DM+HT) %. The flow response to the NO-donor SNP amounted to: 327 (C), 306 (DM), 200 (HT), and 194% (DM+HT). In DM+HT the reduction of endothelium-dependent flow response was more pronounced compared with the endothelium-independent flow response. The present data provide evidence that type 2 diabetes and arterial hypertension impair endothelium-dependent dilation of resistance arteries in an additive manner suggesting that this progressive endothelial dysfunction might contribute to the increased incidence of cardiovascular complications when both diseases are coexistent.

Management of the hypertensive patient with coronary insufficiency but without atherosclerosis.

&NA; Arterial hypertension is a major risk factor for the clinical syndrome of angina pectoris, in which the ECG is abnormal but the coronary arteries are normal. Structural and functional abnormalities in coronary circulation as well as extravascular factors (eg, left‐ventricular hypertrophy, fibrosis with diastolic dysfunction) compromise the adequate ratio of coronary blood flow to oxygen demand causing angina, dyspnea, and major cardiac events. Recent studies stress the importance to functional disturbances of coronary microvasculature leading to profound morphologic changes associated with impaired coronary conductance. In patients without epicardial coronary stenosis hypertensive microvascular disease can be qualitatively assessed by noninvasive diagnostic approaches based on new Doppler echocardiography techniques and may also be monitored by widely available stress tests. For ultimate quantitative assessment, invasive procedures are still required. Beyond guidelines to control blood pressure in hypertensive individuals, restoration of functional and structural integrity of the coronary microvasculature represents the ultimate therapeutic goal in hypertensive patients with coronary insufficiency and without angiographic evidence of atherosclerosis. Concomitant factors reducing coronary conductance such as left‐ventricular hypertrophy and diastolic dysfunction should be reversed in parallel. Currently, therapeutic intervention in the renin‐aldosterone‐angiotensin‐II‐system using ACE inhibitors, angiotensin receptor blockers, and low doses of aldosterone antagonists represent the most promising strategy to achieve these goals. Using the knowledge of these recent results we should refine the overall management of our hypertensive patients with coronary insufficiency but without atherosclerosis.