Arginase II deletion increases corpora cavernosa relaxation in diabetic mice

Abstract

Introduction: Diabetes-induced erectile dysfunction involves elevated arginase (Arg) activity and expression. Because nitric oxide (NO) synthase and Arg share and compete for their substrate L-arginine, NO production is likely linked to regulation of Arg. Arg is highly expressed and implicated in erectile dysfunction.

Aim: It was hypothesized that Arg-II isoform deletion enhances relaxation function of corpora cavernosal (CC) smooth muscle in a streptozotocin (STZ) diabetic model.

Methods: Eight weeks after STZ-induced diabetes, vascular functional studies, Arg activity assay, and protein expression levels of Arg and constitutive NOS (using Western blots) were assessed in CC tissues from nondiabetic wild type (WT), diabetic (D) WT (WT + D), Arg-II knockout (KO), and Arg-II KO+D mice (N = 8-10 per group).

Main outcome measures: Inhibition or lack of arginase results in facilitation of CC relaxation in diabetic CC.

Results: Strips of CC from Arg-II KO mice exhibited an enhanced maximum endothelium-dependent relaxation (from 70 + 3% to 84 + 4%) and increased nitrergic relaxation (by 55%, 71%, 42%, 42%, and 24% for 1, 2, 4, 8 and 16 Hz, respectively) compared with WT mice. WT + D mice showed a significant reduction of endothelium-dependent maximum relaxation (44 + 8%), but this impairment of relaxation was significantly prevented in Arg-II KO+D mice (69 + 4%). Sympathetic-mediated and alpha-adrenergic agent-induced contractile responses also were increased in CC strips from D compared with non-D controls. Contractile responses were significantly lower in Arg-II KO control and D versus the WT groups. WT + D mice increased Arg activity (1.5-fold) and Arg-II protein expression and decreased total and phospho-eNOS at Ser-1177, and nNOS levels. These alterations were not seen in Arg-II KO mice. Additionally, the Arg inhibitor BEC (50 µM) enhanced nitrergic and endothelium-dependent relaxation in CC of WT + D mice.

Conclusion: These studies show for the first time that Arg-II deletion improves CC relaxation in type 1 diabetes.

Figure 1

Endothelium-dependent NO-mediated relaxation to acetylcholine (ACh, 10⁻⁹ to 10⁻⁵ M, panel A) or nitregic nerve stimulation (electric field stimulation, EFS 1–32 Hz, panel B) in cavernosal segments from non-diabetic wild type (WT, open circle), diabetic WT (WT+D, open square), arginase II (Arg-II) knockout (KO, closed circle) and Arg-II KO+D (closed square) mice. Data were calculated relative to the maximal changes from the contraction produced by phenylephrine (PE, 10⁻⁵ M) in each tissue, which was taken as 100%. Data represent the means ± S.E.M. of 8 experiments. *P < 0.05; **P < 0.01, compared with WT mice; ^#P < 0.05; ^!!P < 0.01, compared with WT+D group.

Figure 2

Contractile-response curves upon stimulation of α-₁-adrenergic receptor, phenylephrine (PE, 10⁻⁹ to 10⁻⁴ M, panel A) or adrenergic nerves (electric field stimulation, EFS 1–32 Hz, panel B) in cavernosal segments from non-diabetic wild type (WT, open circle), diabetic WT (WT+D, open square), arginase II (Arg-II) knockout (KO, closed circle) and Arg-II KO+D (closed square) mice. Data were calculated relative to the maximal changes from the contraction produced by KCl (80 mM), which was taken as 100%, and data represent the mean ± S.E.M. of 8–9 experiments. *P < 0.05; **P < 0.01, compared with WT mice; ^#P < 0.05, compared to WT+D mice.

Figure 3

Effect of the arginase inhibitor BEC (5 × 10⁻⁵ M) on the relaxation induced by acetylcholine (ACh, 10⁻⁹ to 3 × 10⁻⁶ M, panel A and C) or on the EFS-induced relaxations (1–32 Hz, panel B and D) in the cavernosal strips from non-diabetic and diabetic in wild type (WT) and Arg-II KO mice. Data were calculated relative to the maximal changes from the contraction produced by phenylephrine (PE, 10⁻⁵ M), which was taken as 100%. Data represent the mean ± S.E.M. of 5–6 experiments. *P < 0.05 and **P < 0.01, compared to WT+D group.

Figure 4

Arginase (Arg) activity in cavernosal tissues from non-diabetic wild type (WT, open bar), diabetic WT (WT+D, grey bar), Arg-II knockout (KO, hatched bar) and Arg-II KO+D (closed bar) mice was determined in corpora cavernosa by urea production. Data are expressed as % of WT. Data represents the mean ± S.E.M. of 5 experiments. *P < 0.05, compared to WT mice.

Figure 5

Western blot analysis of arginase II (Arg-II, panel A) and Arg-I (panel B) in cavernosal tissues of non-diabetic wild type (WT, open bars), diabetic WT (WT+D, grey bars), Arg-II knockout (KO, hatched bars), and Arg-II KO+D (closed bar) mice. A representative blot is shown in the top panel. Results were quantified by densitometry and Arg protein was normalized by α-actin levels expressed as % of WT. Data represents the mean ± S.E.M. of 5 experiments (each group). **P < 0.01, compared to WT mice.

Figure 6

Western blot analysis of total eNOS, nNOS (panel A) and total and phospho eNOS at their regulatory site Ser-1177 (panel B) and Thr-495 (panel C) in cavernosal tissues of non-diabetic wild type (WT, open bar), diabetic WT (WT+D, grey bar), arginase II (Arg-II) knockout (KO, hatched bars) and Arg-II KO+D (closed bars) mice. A representative blot is shown in the top panel. Protein expression of constitutive NOS were normalized by α-actin levels and expressed as % of WT. Data represents the mean ± S.E.M. of 5 experiments (each group). *P < 0.05; **P < 0.01, compared to WT mice.