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. ...

In macrophages and many other cell types,l-arginine is used as a substrate by both nitric oxide synthase (NOS) and arginase to produce nitric oxide (NO) and urea, respectively. Because the availability ofl-arginine is a major determinant for NO synthesis in the activated macrophage, we hypothesized that NO production may be reduced by arginase via depleting the common substrate in this cell type. To test this hypothesis, we investigated the effect of an arginase inhibitor,l-norvaline, on NO production in J774A.1 mouse macrophages activated by lipopolysaccharide (LPS, 1.0 μg/ml) for 22 h. In the absence of LPS, macrophages produced a low level of NO. In contrast, NO production from these cells was significantly increased in the presence of LPS. Increasing extracellular levels ofl-arginine (0.01–0.8 mM) produced a concomitant increase in NO production of activated macrophages. l-Norvaline (10 mM), which specifically inhibits arginase activity (i.e., reducing urea production by 50%) without altering NOS activity, enhanced NO production (by 55%) from activated macrophages. The enhancement of NO production by l-norvaline was inversely related to the extracellular level ofl-arginine. A more pronounced increase in NO production was observed at the lower level of extracellular l-arginine, i.e., a 55 vs. 28% increase for 0.05 and 0.1 mM extracellularl-arginine, respectively. When the l-arginine concentration exceeded 0.5 mM, thel-norvaline effect was abolished. These results indicate that arginase can compete with NOS for their common substrate and thus inhibit NO production. This regulatory mechanism may be particularly important when the extracellular supply ofl-arginine is limited.

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.

In macrophages, l-arginine can be used by NO synthase and arginase to form NO and urea, respectively. Therefore, activation of arginase may be an effective mechanism for regulating NO production in macrophages through substrate competition. Here, we examined whether IL-13 up-regulates arginase and thus reduces NO production from LPS-activated macrophages. The signaling molecules involved in IL-13-induced arginase activation were also determined. Results showed that IL-13 increased arginase activity through de novo synthesis of the arginase I mRNA and protein. The activation of arginase was preceded by a transient increase in intracellular cAMP, tyrosine kinase phosphorylation, and p38 mitogen-activated protein kinase (MAPK) activation. Exogenous cAMP also increased arginase activity and enhanced the effect of IL-13 on arginase induction. The induction of arginase was abolished by a protein kinase A (PKA) inhibitor, KT5720, and was down-regulated by tyrosine kinase inhibitors and a p38 MAPK inhibitor, SB203580. However, inhibition of p38 MAPK had no effect on either the IL-13-increased intracellular cAMP or the exogenous cAMP-induced arginase activation, suggesting that p38 MAPK signaling is parallel to the cAMP/PKA pathway. Furthermore, the induction of arginase was insensitive to the protein kinase C and p44/p42 MAPK kinase inhibitors. Finally, IL-13 significantly inhibited NO production from LPS-activated macrophages, and this effect was reversed by an arginase inhibitor, l-norvaline. Together, these data demonstrate for the first time that IL-13 down-regulates NO production through arginase induction via cAMP/PKA, tyrosine kinase, and p38 MAPK signalings and underline the importance of arginase in the immunosuppressive activity of IL-13 in activated macrophages.