Constitutive expression of arginase in microvascular endothelial cells counteracts nitric oxide‐mediated vasodilatory function

Abstract

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. ...

Arginase modulates myocardial contractility by a nitric oxide synthase 1-dependent mechanism

“…Arg I and Arg II expression has also been demonstrated in the penis (11,16) and A293 cells overexpressing NOS1 (44), where reciprocal regulation of Arg and constitutive NOS1 exists. Our published data (10) and those of other researchers (20,45,46) support the notion that Arg isoforms are expressed constitutively in vascular endothelium and may (as in the airway, the penis, and A293 cells) modulate NOS activity by regulating L-arginine availability.…”

“…Whereas Arg II is expressed exclusively in the cardiac myocytes, both Arg I and II are found in whole-heart homogenates. These findings most likely reflect the Arg that is present in cell types other than myocytes, such as endothelial cells that have been shown to express Arg I (10,20). Consistent with the data from WB, immunostaining demonstrated Arg II but not Arg I in isolated myocytes (Fig.…”

“…Rat hearts and myocytes were homogenized in lysis buffer (50 mM Tris⅐HCl, pH 7.5͞0.1 mM EDTA, with protease inhibitor) and centrifuged for 30 min at 14,000 ϫ g at 4°C for an Arg-activity assay, as described (20).…”

Developmental changes in arginase expression and activity in the lung

“…Previously, Berkowitz et al (6) had shown that both arginase isotypes were expressed in the rat aortic endothelium. Arginase I had been found to be constitutively expressed in endothelial cells (35), whereas arginase II appeared to be inducible in response to cytokines (8). White et al (32) have demonstrated that arginase type I knockdown could enhance NOS activity in adult rat thoracic aortic tissue, and Zhang et al (35) had shown in the porcine coronary artery that arginase type I is responsible for modulating the endothelial-dependent relaxation.…”

“…Arginase I had been found to be constitutively expressed in endothelial cells (35), whereas arginase II appeared to be inducible in response to cytokines (8). White et al (32) have demonstrated that arginase type I knockdown could enhance NOS activity in adult rat thoracic aortic tissue, and Zhang et al (35) had shown in the porcine coronary artery that arginase type I is responsible for modulating the endothelial-dependent relaxation. Bachetti et al (4) demonstrated that both isoforms were expressed in human umbilical vein endothelial cells (HUVEC) with a predominance of the type I isoform.…”