Endothelial arginine resynthesis contributes to the maintenance of vasomotor function in male diabetic mice

Abstract

Aim: Argininosuccinate synthetase (ASS) is essential for recycling L-citrulline, the by-product of NO synthase (NOS), to the NOS substrate L-arginine. Here, we assessed whether disturbed arginine resynthesis modulates endothelium-dependent vasodilatation in normal and diabetic male mice.

Methods and results: Endothelium-selective Ass-deficient mice (Assfl/fl/Tie2Cretg/- = Ass-KOTie2) were generated by crossing Assfl/fl mice ( = control) with Tie2Cre mice. Gene ablation in endothelial cells was confirmed by immunohistochemistry. Blood pressure (MAP) was recorded in 34-week-old male mice. Vasomotor responses were studied in isolated saphenous arteries of 12- and 34-week-old Ass-KOTie2 and control animals. At the age of 10 weeks, diabetes was induced in control and Ass-KOTie2 mice by streptozotocin injections. Vasomotor responses of diabetic animals were studied 10 weeks later. MAP was similar in control and Ass-KOTie2 mice. Depletion of circulating L-arginine by arginase 1 infusion or inhibition of NOS activity with L-NAME resulted in an increased MAP (10 and 30 mmHg, respectively) in control and Ass-KOTie2 mice. Optimal arterial diameter, contractile responses to phenylephrine, and relaxing responses to acetylcholine and sodium nitroprusside were similar in healthy control and Ass-KOTie2 mice. However, in diabetic Ass-KOTie2 mice, relaxation responses to acetylcholine and endothelium-derived NO (EDNO) were significantly reduced when compared to diabetic control mice.

Conclusions: Absence of endothelial citrulline recycling to arginine did not affect blood pressure and systemic arterial vasomotor responses in healthy mice. EDNO-mediated vasodilatation was significantly more impaired in diabetic Ass-KOTie2 than in control mice demonstrating that endothelial arginine recycling becomes a limiting endothelial function in diabetes.

Figure 1. Expression of ASS protein in saphenous arteries of male control (panel A) and Ass-KO^Tie2 (panel C) mice.

The arrow indicates endothelial expression in control animals (panel A) and absence of endothelial expression in Ass-KO^Tie2 mice (panel C). Panels B and D represent the corresponding H&E staining of a serial section to demonstrate the presence of intact endothelium.

Figure 2. The effect of endothelium-specific Ass deletion on hemodynamics of 34-week-old conscious male mice.

Black bar: control mice; white bar: Ass-KO^Tie2 mice. Blood pressure was measured in the same mice 2 (panel A) and 3 days (panel B) after catheterization via a femoral artery catheter connected to a pressure transducer. Panel A: mean arterial pressure (MAP) in the basal condition (left) and after a bolus infusion of 200 U bovine arginase 1 via a jugular vein catheter (right). Panel B: mean arterial pressure in the basal condition (left) and after intravenous L-NAME (10 mg/kg) infusion (right). Values are means ± SEM (control animals: arginase 1: n = 7, L-NAME: n = 5; Ass-KO^Tie2 mice: arginase 1: n = 5, L-NAME: n = 4; due to loss of catheter patency, numbers were lower on the 3^rd day). Note that the Y-axis starts at 90 mm Hg.

Figure 3. The effect of endothelium-specific Ass deletion on relaxation responses of saphenous arteries of healthy and diabetic male mice.

Relaxation of PHE (10 µM)-pre-contracted saphenous arteries of 12- (panels A–C) and 34-week-old (panels D–F) healthy and 22-week-old diabetic (panels G–I) male mice to ACh (0.01–10 µM) was determined by wire myography. Black squares: control mice; white circles: Ass-KO^Tie2 mice. Panels (A, D, G): relaxation responses in the absence of pharmacological inhibitors. Panels (B, E, H): relaxation responses in the presence of INDO (10 µM). Panels (C, F, I): relaxation responses in the presence of both INDO (10 µM) and L-NAME (100 µM). Values are shown as means ± SEM (n = 5–7; for the number of animals per individual experiment see Table 1). *P<0.05 vs. the control, **P<0.01 vs. the control (unpaired t-test).

Figure 4. The effect of endothelium-specific Ass deletion on relaxation responses of saphenous arteries of healthy and diabetic male mice.

Relaxation of K⁺ (40 mM)-pre-contracted saphenous arteries of 12- (panel A) and 34-week-old (panel B) healthy and 22-week-old diabetic (panel C) male mice to ACh (0.01–10 µM) was determined by wire myography. Black squares: control mice; white circles: Ass-KO^Tie2. All arteries were treated with INDO (10 µM). Values are shown as means ± SEM (n = 4–8; for the number of animals per individual experiment, see Table 1). **P<0.01 vs. control (unpaired t-test).

Figure 5. The effect of endothelium-specific Ass deletion on relaxation responses of saphenous arteries to sodium nitroprusside.

Relaxation of PHE pre-contracted (10 µM) saphenous arteries of 12- (panel A) and 34-week-old healthy (panel B) and 22-week-old diabetic (C) male mice to SNP (0.01–10 µM) was determined by wire myography. Black squares: control mice; white circles: Ass-KO^Tie2. All experiments were performed in the presence of L-NAME (100 µM) and INDO (10 µM). Values are means ± SEM (n = 5–7; for the number of animals per individual experiment, see Table 1).