Arginase 1 contributes to diminished coronary arteriolar dilation in patients with diabetes

Abstract

Arginase 1, via competing with nitric oxide (NO) synthase for the substrate L-arginine, may interfere with NO-mediated vascular responses. We tested the hypothesis that arginase 1 contributes to coronary vasomotor dysfunction in patients with diabetes mellitus (DM). Coronary arterioles were dissected from the right atrial appendages of 41 consecutive patients with or without DM (the 2 groups suffered from similar comorbidities), and agonist-induced changes in diameter were measured with videomicroscopy. We found that the endothelium-dependent agonist ACh elicited a diminished vasodilation and caused constriction to the highest ACh concentration (0.1 μM) with a similar magnitude in patients with (18 ± 8%) and without (17 ± 9%) DM. Responses to ACh were not significantly affected by the inhibition of NO synthesis with N(G)-nitro-L-arginine methyl ester in either group. The NO donor sodium nitroprusside-dependent dilations were not different in patients with or without DM. Interestingly, we found that the presence of N(G)-hydroxy-L-arginine (10 μM), a selective inhibitor of arginase or application of L-arginine (3 mM), restored ACh-induced coronary dilations only in patients with DM (to 47 ± 6% and to 40 ± 19%, respectively) but not in subjects without DM. Correspondingly, the protein expression of arginase 1 was increased in coronary arterioles of patients with DM compared with subjects without diabetes. Moreover, using immunocytochemistry, we detected an abundant immunostaining of arginase 1 in coronary endothelial cells of patients with DM, which was colocalized with NO synthase. Collectively, we provided evidence for a distinct upregulation of arginase 1 in coronary arterioles of patients with DM, which contributes to a reduced NO production and consequently diminished vasodilation.

Fig. 1.

Changes in diameter of coronary arterioles isolated from patients without diabetes mellitus (DM−, n = 11) and patients with DM (DM+, n = 10) in response to cumulative concentrations of ACh in the absence (A) or in the presence (B, n = 6 in each group) of nitric oxide synthesis inhibitor N^G-nitro-l-arginine methyl ester (l-NAME). Changes in diameter of coronary arterioles isolated from subjects without (DM−, n = 9) and with diabetes (DM+, n = 8) in response to cumulative concentrations of sodium nitroprusside (SNP; C) are shown. Data are means ± SE. *P < 0.05, significant changes in diameter from the baseline diameters on A and B.

Fig. 2.

Changes in diameter of coronary arterioles isolated from patients without (DM−) and with (DM+) diabetes in response to cumulative concentrations of ACh before and after incubation with N^G-hydroxy-l-arginine (l-NOHA, n = 5–5; A) or with l-arginine (l-Arg, n = 6–6; B). Data are means ± SE. *P < 0.05, significant differences in the diameter changes before and after incubation with l-NOHA or l-Arg.

Fig. 3.

A, top: Western blot analysis of the expression of arginase 1 (Arg 1) in coronary arterioles isolated from the atrial appendage of patients with (n = 5) and without (n = 5) DM. Anti-β-actin was used to normalize for loading variations. A, bottom: summary of normalized densitometric ratios. Data are means ± SE. B: representative images of individual endothelial cells (×100, 1.4 numerical aperature objective) on acetone-fixed, en face coronary arteries obtained from patients without (DM−, n = 4; left) or with (DM+, n = 4; right) DM. Cells were immunolabeled with primary antibodies against endothelial nitric oxide synthase (eNOS) and Arg 1. Fluorescent labeling of anti-eNOS was performed with Alexa 488 secondary antibody (shown in green) and Alexa 597 antibody (shown in red) for anti-Arg 1. 4,6-Diamidino-2-phenylindole (DAPI) was used for nuclear staining (shown in blue). Coimmunostaining is shown in orange/yellow in the merged images; cells represent 5 cells from 3 different regions in each patient.