Arginase shares a common substrate, L-arginine, with nitric oxide (NO) synthase (NOS). It is thought that arginase, if it is expressed in the endothelium, might play a pivotal role in the regulation of NO-mediated vasodilation by reducing the L-arginine availability to NOS. In the present study, we wanted to determine whether arginase is expressed and active in coronary arterioles and to demonstrate whether endothelial arginase can influence NO production and play a functional role in regulating NO-mediated dilation. In this regard, the expression of arginase mRNA and distribution of arginase protein in porcine coronary microvessels were determined by reverse transcription-polymerase chain reaction and immunohistochemistry, respectively. To assess the role of arginase in vasoregulation directly, porcine subepicardial coronary arterioles (60-110 µm in diameter) were isolated, cannulated, and pressurized for in vitro study under the conditions with and without arginase inhibition. Molecular evidence indicated that arginase I, but not arginase II, mRNA was expressed in the coronary arterioles. The constitutive expression of arginase I protein in the coronary arteriolar endothelial cells was revealed immunohistochemically. Adenosine and serotonin stimulated a threefold increase in NO release and produced dilation of isolated coronary arterioles. NOS inhibitor L-NMMA abolished the stimulated NO release and attenuated the dilations in response to these agonists. In contrast to L-NMMA, arginase inhibitor α-difluoromethylornithine (DFMO) increased the NO release by about 80% and also enhanced vasodilations in response to adenosine and serotonin. DFMO inhibited arginase (by 80%), but not NOS, activity in these microvessels without affecting their dilation in response to sodium nitroprusside. Similar to DFMO, intraluminal application of L-arginine enhanced NO-mediated vasodilations. The DFMO-enhanced vasodilation was not observed in the presence of L-NMMA or after endothelial removal, which suggests a regulatory role of endothelial arginase in the NOmediated response. Collectively, this study provides novel findings that the arginase expressed in the endothelium plays a counteracting role in the stimulated NO production, and thus NO-mediated vasodilatory function.Key Words: coronary microcirculation • RT-PCR • immunohistochemistry -arginine is the substrate for both nitric oxide (NO) synthase (NOS) and arginase (1, 2). Thus far, three distinct NOS isoforms have been isolated and purified (3). Each of these enzymes is involved in the catabolism of l-arginine to form NO and citrulline (4,5). NO is a major messenger molecule that has been shown to regulate blood vessel dilation and immune function and to serve as a neurotransmitter in the brain and peripheral nervous system (6). In endothelial cells, the regulated control of a constitutive NOS allows for the maintenance of vascular tone and normal blood pressure, and it inhibits platelet adherence and aggregation.A second major pathway of arginine metabolism is via arginase. ...

Nitric oxide (NO) produced by the endothelium diffuses both into the lumen and to the smooth muscle cells according to the concentration gradient in each direction. The extremely high reaction rate between NO and hemoglobin (Hb), k Hb ‫؍‬ 3-5 ؋ 10 7 M ؊1 ⅐s ؊1 , suggests that most of the NO produced would be consumed by Hb in the red blood cells (RBCs), which then would block the biological effect of NO. Therefore, specific mechanisms must exist under physiological conditions to reduce the NO consumption by RBCs, in which the Hb concentration is very high (24 mM heme). By using isolated microvessels as a bioassay, here we show that physiological concentrations of RBCs in the presence of intravascular f low does not inhibit NO-mediated vessel dilation, suggesting that RBCs under this condition are not an NO scavenger. On the other hand, RBCs (50% hematocrit) without intravascular f low reduce NO-mediated dilation to serotonin by 30%. In contrast, free Hb (10 M) completely inhibits NO-mediated dilation with or without intravascular f low. The effect of f low on NO consumption by RBCs may be attributed to the formation of an RBC-free zone near the vessel wall, which is caused by hydrodynamic forces on particles. Intravascular f low does not affect the reaction rate between NO and free Hb in the lumen, because the latter forms a homogeneous solution and is not subject to the hydrodynamic separation. However, intravascular f low only partially contributes to the reduced consumption of NO by RBCs, because without the f low, the NO consumption by RBCs is already about 3 orders of magnitude slower than free Hb.Nitric oxide (NO) is a biological messenger that participates in neurotransmission, vascular regulation, and immunological responses. It is produced in many cell types such as neurons, endothelial cells vascular smooth muscle cells, and macrophages. Since the discovery of its biological activity, the roles of NO in physiological and pathophysiological processes have been seen as increasingly important. In the cardiovascular system, NO has been documented to participate in the regulation of vascular tone and permeability (1, 2), platelet adhesion and aggregation (3, 4), smooth muscle proliferation (5, 6), and endothelial cell-leukocyte interactions (1, 7). The functions of NO in vivo have been clearly demonstrated by administration of L-arginine analogs that block NO production. For example, the administration of N G -monomethyl-L-arginine (L-NMMA) has been shown to cause hypertension in vivo (8) and abolish shear-induced, NO-mediated vessel dilation both in vivo (9) and in isolated intact vessel preparation (10).Despite the well-documented importance of NO, the transfer of NO from the producing cell to the target is poorly understood, because NO, as a free radical, can be degraded in a variety of reactions. In particular, NO reacts with deoxy-and oxy-hemoglobin (Hb) at a very high rate to form nitrosyl-Hb (HbNO), and metHb, respectively. The bimolecular reaction rate constants for these reactions are on th...

A reduction in L-arginine availability has been implicated in the impairment of endothelium-dependent nitric oxide (NO)-mediated vasodilation by ischemia-reperfusion (I/R). However, the mechanisms contributing to dysregulation of the L-arginine pool remain unknown. Because endothelial cells can metabolize L-arginine via two major enzymes, that is, NO synthase (NOS) and arginase, we hypothesized that up-regulation of arginase during I/R reduces L-arginine availability to NOS and thus impairs NO-mediated vasodilation. To test this hypothesis, a local I/R was produced in the porcine heart by occlusion of a small branch of left anterior descending artery for 30 min, followed by reperfusion for 90 min. Arterioles (60-110 microm) isolated from non-ischemic and ischemic regions of subepicardium were cannulated and pressurized without flow for in vitro study. Vessels from both regions developed similar levels of basal tone. Although the dilation of I/R vessels to endothelium-independent agonist sodium nitroprusside was not altered, the endothelium-dependent NO-mediated dilations to adenosine and serotonin were attenuated. I/R not only inhibited arteriolar production of NO but also increased arteriolar arginase activity. Arginase inhibitor alpha-difluoromethylornithine enhanced NO production/dilation in normal vessels and also restored the NO-mediated function in I/R vessels. Treating I/R vessels with L-arginine also restored vasodilations. Immunohistochemical data revealed that I/R up-regulated arginase but down-regulated NOS expression in the arteriolar endothelium. Pretreating the animals with protein synthesis inhibitor cycloheximide prevented I/R-induced arginase up-regulation and also preserved NO-mediated vascular function. These results suggest that one mechanism by which I/R inhibits NO-mediated arteriolar dilation is through increased arginase activity, which limits the availability of L-arginine to NOS for NO production. In addition, the inability of arginase blockade or L-arginine supplementation to completely restore vasodilatory function may be attributable to the down-regulation of endothelial NOS expression.

Abstract-One characteristic of hypertension is a decreased endothelium-dependent nitric oxide (NO)-mediated vasodilation; however, the underlying mechanism is complex. In endothelial cells (ECs), L-arginine is the substrate for both NO synthase (NOS) and arginase. Because arginase has recently been shown to modulate NO-mediated dilation of coronary arterioles by reducing L-arginine availability, we hypothesized that upregulation of vascular arginase in hypertension contributes to decreased NO-mediated vasodilation. To test this hypothesis, hypertension (mean arterial blood pressure Ͼ150 mm Hg) was maintained for 8 weeks in pigs by aortic coarctation. Coronary arterioles from normotensive (NT) and hypertensive (HT) pigs were isolated and pressurized for in vitro study. NT vessels dilated dose-dependently to adenosine (partially mediated by endothelial release of NO) and sodium nitroprusside (endothelium-independent vasodilator). Conversely, HT vessels exhibited reduced dilation to adenosine but dilated normally to sodium nitroprusside. Adenosine-stimulated NO release was increased Ϸ3-fold in NT vessels but was reduced in HT vessels. Moreover, arginase activity was 2-fold higher in HT vessels. Inhibition of arginase activity by N -hydroxynor-L-arginine or incubation with L-arginine partially restored NO release and dilation to adenosine in HT vessels. Immunohistochemistry showed that arginase expression was increased but NOS expression was decreased in arteriolar ECs of HT vessels. These results suggest that NO-mediated dilation of coronary arterioles is inhibited in hypertension by an increase in arginase activity in EC, which limits L-arginine availability to NOS for NO production. The inability of arginase blockade or L-arginine supplementation to completely restore vasodilation may be related to downregulation of endothelial NOS expression. Key Words: arginine Ⅲ hypertension Ⅲ microcirculation Ⅲ nitric oxide synthase Ⅲ vasodilation H ypertension is a major risk factor for coronary artery disease. One characteristic of hypertension that appears to be critical in the development of vascular disease is the impairment of endothelial function. For example, there is a markedly reduced endothelium-dependent vasodilation in both large and small coronary arteries from hypertensive humans and animal models of hypertension. 1 Mounting evidence suggests that this vascular impairment may be related to a diminished production and bioavailability of the potent vasodilator nitric oxide (NO), which may result from an increased vascular production of superoxide anion 2 or decreased endothelial levels of tetrahydrobiopterin 3,4 or L-arginine. 5 Interestingly, administration of NO precursor L-arginine restores endothelium-mediated vasodilatory function in patients with essential hypertension, 5 improves coronary hemodynamics in spontaneously hypertensive rats, 6 and increases NO production and reduces blood pressure in hypertensive rats with 7 or without 8 renal failure. These results suggest the possible reduction of L-arginine ...

Nitric oxide (NO) activates soluble guanylyl cyclase in smooth muscle cells to induce vasodilation in the vasculature. However, as hemoglobin (Hb) is an effective scavenger of NO and is present in high concentrations inside the red blood cell (RBC), the bioavailability of NO would be too low to elicit soluble guanylyl cyclase activation in the presence of blood. Therefore, NO bioactivity must be preserved. Here we present evidence suggesting that the RBC participates in the preservation of NO bioactivity by reducing NO influx. The NO uptake by RBCs was increased and decreased by altering the degree of band 3 binding to the cytoskeleton. Methemoglobin and denatured hemoglobin binding to the RBC membrane or cytoskeleton also were shown to contribute to reducing the NO uptake rate of the RBC. These alterations in NO uptake by the RBC, hence the NO bioavailability, were determined to correlate with the vasodilation of isolated blood vessels. Our observations suggest that RBC membrane and cytoskeleton associated NO-inert proteins provide a barrier for NO diffusion and thus account for the reduction in the NO uptake rate of RBCs. E xtensive studies have established the bioactivity of nitric oxide (NO) in vasoregulation through activation of soluble guanylyl cyclase (ref. 1 and references therein). On the other hand, NO is rapidly deactivated by oxygenated hemoglobin (HbO 2 ) and myoglobin (MbO 2 ) to form nitrate. As NO reacts rapidly (k ϳ 10 7 M Ϫ1 ⅐s Ϫ1) with HbO 2 and deoxygenated Hb (deoxyHb), infusion of cell-free normoxic Hb (hereafter, Hb denotes both HbO 2 and deoxyHb) at M levels into animal models, human subjects, or isolated blood vessels (2-5) causes significant vessel constriction due to NO scavenging. However, normal blood containing RBC-encapsulated Hb (rbcHb) at an equivalent concentration of about 10 mM shows insignificant NO reactivity under physiological conditions (4,6,7). This discrepancy is difficult to explain in view of the high permeability of NO through lipid bilayers. Thus, NO bioactivity in blood must be preserved by a yet unclear mechanism under physiological conditions. This problem recently was addressed by a NO bioactivity export theory (8). According to this theory, NO enters the RBC and preferentially binds with the free heme on Hb to form heme-nitrosylHb (HbNO) rather than being oxidized by O 2 -conjugated heme (9). HbNO then transfers the conjugated NO to ␤-93Cys to form S-nitrosoHb (9). NO bioactivity is then exported out of RBCs through the anion exchange protein, band 3 (or AE1). Although detailed mechanisms of SNO (Snitrosothiol species) formation and NO bioactivity export from band 3 are still unclear (10, 11), the theory provides an explanation for the preservation of NO bioactivity and highlights the importance of the RBC in preserving NO bioavailability, which has been suggested previously (12).In parallel to the NO bioactivity export theory, an independent, but not mutually exclusive, explanation that NO bioavailability is preserved by reducing interactions between NO and r...

Objective-Elevated levels of C-reactive protein (CRP), a proinflammatory marker, are associated with reduced systemic endothelium-dependent NO-mediated dilation in patients with coronary artery disease; however, the direct effect of CRP on coronary microvascular reactivity remains unknown. Herein, we examined whether CRP can modulate endothelium-dependent NO-mediated dilation of coronary arterioles and whether proinflammatory signaling pathways such as stress-activated protein kinases (p38 and c-Jun N-terminal kinase [JNK]) and oxidative stress are involved in the CRP-mediated effect. Methods and Results-Porcine coronary arterioles were isolated and pressurized without flow for in vitro study.Intraluminal treatment with a clinically relevant concentration of CRP (7 g/mL; 1 hour) significantly attenuated the NO release and vasodilation to serotonin. Further incubation with the NO precursor L-arginine (3 mmol/L) partially restored serotonin-induced vasodilation. In the presence of superoxide scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), NAD(P)H oxidase inhibitor apocynin, or p38 kinase (an upstream activator of NAD(P)H oxidase) inhibitor SB203850, but not xanthine oxidase inhibitor allopurinol or JNK inhibitor SP600125, the detrimental effect of CRP on serotonin-induced dilation was prevented. Dihydroethidium staining showed that CRP produced SB203850-and TEMPOL-sensitive superoxide production in the arteriolar endothelium. CRP treatment of coronary arterioles significantly increased NAD(P)H oxidase activity. Conclusions-CRP inhibits endothelium-dependent NO-mediated dilation in coronary arterioles by producing superoxide from NAD(P)H oxidase via p38 kinase activation. By impairing endothelium-dependent NO-mediated vasoreactivity, CRP could facilitate the initiation of numerous cardiovascular diseases. Key Words: C-reactive protein Ⅲ nitric oxide Ⅲ free radicals Ⅲ coronary artery disease A growing wealth of evidence supports the paradigm that inflammation plays a pivotal role in the development and progression of atherosclerosis. 1 C-reactive protein (CRP) is an acute-phase inflammatory marker that has been shown in several prospective studies to be an independent risk factor for cardiovascular events such as stroke, myocardial infarction, and coronary artery disease. 2-4 Recent evidence suggests that CRP may not only be a marker but also a mediator of inflammation and atherogenesis via direct effects on leukocytes and vascular cells. For example, CRP promotes monocyte chemotaxis 5 and facilitates low-density lipoprotein uptake by macrophages in vitro. 6 In vascular smooth muscle cells, CRP has been shown to increase angiotensin type 1 receptor number and angiotensin type 1 receptor-mediated reactive oxygen species formation, 7 as well as activation of stress-activated protein kinases p38 kinase and c-Jun N-terminal kinase (JNK). 8 In endothelial cells, CRP facilitated the release of plasminogen activator inhibitor-1 9 and endothelin-1, 10 increased the expression of cell adhesion molecules, 1...

Abstract-Angiotensin II (Ang II) is a potent vasoconstrictor in the peripheral circulation and has been implicated in many cardiovascular diseases associated with elevated oxidative stress. However, its direct vasomotor action and its linkage to oxidative stress-induced vascular dysfunction in the coronary microcirculation remain elusive. In this study, we directly assessed the vasomotor action of Ang II in isolated porcine coronary arterioles and also examined whether Ang II can modulate endothelium-dependent nitric oxide (NO)-mediated dilation via superoxide production. Ang II evoked vasoconstriction at a low concentration (1 nmol/L) and dilations at higher concentrations (Ͼ10 nmol/L). Ang II type 1 (AT 1 ) receptor antagonist losartan abolished vasoconstriction, whereas Ang II type 2 (AT 2 ) receptor antagonist PD 123319 eliminated vasodilation. Adenosine stimulated a significant arteriolar NO production and dilation. NO synthase inhibitor N G -monomethyl-L-arginine (L-NMMA) abolished stimulated NO production and attenuated vasodilation. Pretreating vessels with a subvasomotor concentration of Ang II (0.1 nmol/L, 60 minutes) mimicked inhibitory effects of L-NMMA. Ang II-mediated inhibition was not observed in the presence of L-NMMA or after endothelial removal but was prevented by losartan, superoxide scavenger TEMPOL, or NADPH oxidase inhibitor apocynin. Dihydroethidium staining showed that Ang II elicited losartan-and TEMPOL-sensitive superoxide production in arterioles. These results demonstrate that Ang II evokes AT 1 receptor-mediated vasoconstriction and AT 2 receptor-mediated vasodilation of coronary arterioles. Ang II at a subvasomotor level impairs endothelium-dependent NO-mediated dilation attributable to elevated superoxide production via AT 1 receptor activation of NADPH oxidase. These data may partly explain the impaired coronary flow regulation in heart diseases associated with an upregulated renin-angiotensin system. (Circ Res. 2003;92:322-329.)

Background and Objective-Elevated C-reactive protein (CRP) levels are associated with increased cardiovascular events and endothelial dysfunction. We have previously shown that CRP decreases endothelial nitric oxide synthase (eNOS) activity in endothelial cells and inhibits endothelium-dependent nitric oxide (NO)-mediated vasodilation in-vitro. Herein, we examined the effect of in-vivo administration of CRP on endothelial function and underlying mechanisms in a valid animal model.