Involvement of accumulated endogenous NOS inhibitors and decreased NOS activity in the impaired neurogenic relaxation of the rabbit proximal urethra with ischaemia

Abstract

1. We examined the effect of ischaemia on the neurogenic and nitric oxide (NO)-mediated urethral relaxation. 2. Rabbits were divided into control and urethral ischaemia (UI) groups, which was prepared by the partial occlusion of bilateral iliac arteries using blood vessel occluders. 3. Neurogenic and NO-mediated proximal urethral relaxation induced by electrical field stimulation (EFS) was greatly impaired in the UI group, while relaxation by sodium nitroprusside (SNP) as a NO donor showed no difference between the two groups. Pretreatment with L-arginine significantly improved but did not normalize the impaired relaxation in the UI group. Not only basal level, but also stimulated production of cyclic GMP with EFS, were significantly decreased in the UI group. 4. The tissue contents of N(G)-methyl-L-arginine (L-NMA) and asymmetric N(G), N(G)-dimethyl-L-arginine (ADMA) in the proximal urethra were increased following ischaemia. While L-arginine and symmetric N(G), N'(G)-dimethyl-L-arginine (SDMA) contents remained unchanged. Exogenously applied authentic L-NMA and ADMA (1 -- 100 microM) concentration-dependently inhibited the EFS-induced urethral relaxation in the control group. The inhibition with L-NMA and ADMA was undetectable in the presence of 3 mM L-arginine. 5. The Ca(2+)-dependent NOS activity in the urethra from the UI group was significantly lower than that from the control group and was not restored by an addition of 3 mM L-arginine. 6. These results suggest that the impaired neurogenic and NO-mediated urethral relaxation with ischaemia is closely related to the increased accumulation of L-NMA and ADMA and decreased NOS activity, which would result in an accelerated reduction in NO production/release.

Figure 1

Representative tracings showing relaxation responses to electrical field stimulation (EFS) and sodium nitroprusside (SNP) in rabbit urethral strips from control (a) and urethral ischaemia (UI, b) groups and the effects of ADMA and tetrodotoxin (TTX). EFS were delivered to strips precontracted with phenylephrine (PE, 10 μM) in the presence of atropine (1 μM) and guanethidine (10 μM). In the control group (a), 100 μM ADMA partially inhibited the EFS induced relaxation. Addition of tetrodotoxin (TTX, 1 μM) abolished the responses to EFS in both groups.

Figure 2

Electrical field stimulation (EFS)-induced relaxation during the contraction caused by phenylephrine (PE, 10 μM) in rabbit urethral strips of control and urethral ischaemia (UI) groups and effects of N^G-nitro-L-arginine (L-NOARG, 100 μM) and L-arginine (3 mM). Atropine (1 μM) and guanethidine (10 μM) were present throughout the experiments. Results are expressed as a percentage of the PE contraction. Data points represent mean±s.e.mean of measurements in 5 – 7 strips from different animals. Vertical bars show s.e.mean. ^aP<0.05; ^bP<0.01 (ANOVA) vs frequency-response curve obtained in the untreated control group. ^cP<0.05; ^dP<0.01 (ANOVA) vs the curve obtained in the untreated UI group.

Figure 3

Sodium nitroprusside (SNP)-induced relaxation during the contraction caused by PE (10 μM) in rabbit urethral strips of control and UI groups and effect of ODQ (10 μM). Atropine (1 μM) and guanethidine (10 μM) were present throughout the experiments. SNP was added cumulatively after the PE contraction had reached a steady-state level. Results are expressed as a percentage of the PE contraction. Data points represent mean±s.e.mean of measurements in 5 – 7 strips from different animals. Vertical bars show s.e.mean. ^bP<0.01 (ANOVA) vs concentration-response curve obtained in the untreated control group. ^dP<0.01 (ANOVA) vs the curve obtained in the untreated UI group.

Figure 4

Cyclic GMP levels measured under basal condition and electrical field stimulation (EFS) in the control and urethral ischaemia (UI) rabbits. Results are given as mean±s.e.mean of 5 – 7 determinations from different animals. Vertical bars show s.e.mean. ^a and ^b; significantly different from the control at P<0.05 and P<0.01, respectively. All experiments were performed in the presence of 10 μM 3-isobutyl-l-methylxanthine (IBMX) as a non-selective phosphodiesterase inhibitor. The net production of cyclic GMP was expressed as the difference between the production stimulated by EFS and that stimulated by EFS in the presence of N^G-nitro-L-arginine (L-NOARG, 100 μM) as an inhibitor of nitric oxide synthase. The basal level was given as the value without EFS.

Figure 5

Inhibitory effects of L-NMA and ADMA on the urethral relaxations induced by EFS in the control (a) and urethral ischaemia (UI) (b) groups. The strips were pretreated with L-NMA or ADMA for 30 min. Atropine (1 μM) and guanethidine (10 μM) were present throughout the experiments. Results are expressed as a percentage of the PE (10 μM) contraction. Data points represent mean±s.e.mean of measurements in 5 – 7 strips from different animals. Vertical bars show s.e.mean. ^aP<0.05; ^bP<0.01 (ANOVA) vs frequency-response curve obtained in the untreated control group. ^cP<0.05 (ANOVA) vs curve obtained in the untreated UI group.

Figure 6

NOS activity in the proximal urethra in the control and UI groups. NOS activity was measured in the presence of 2 mM CaCl₂ (Ca²⁺), in the absence of CaCl₂ and in the presence of 2 mM EGTA (-Ca²⁺) and in the presence of CaCl₂ and 100 μM N^G-nitro-L-arginine (L-NOARG). All reaction mixtures contained 2 mM NADPH, 30 U ml⁻¹ calmodulin, 5 μM FAD, 14 μM BH₄ and 20 μM L-arginine. Data represent mean±s.e.mean of five measurements from different animals. Vertical bars show s.e.mean. ^aP<0.05, ^bP<0.01 vs untreated control, ^dP<0.01 vs untreated UI.

Figure 7

Representative experiment showing the plot of velocity for NOS activity (pmol citrulline mg⁻¹ protein min⁻¹) vs concentration of L-arginine in the proximal urethra in the control and UI groups. NOS activity was measured in the presence of 2 mM CaCl₂, 2 mM NADPH, 30 U ml⁻¹ calmodulin, 5 μM FAD, 14 μM BH₄ at different L-arginine concentrations (0 – 3000 μM). The K_M and V_max values were estimated by non-linear regression, fitting the data to a rectangular hyperbola.

Figure 8

Inhibitory effects of L-NOARG, L-NMA and ADMA on the NOS activity in the control (a) and UI (b) groups. Values are expressed as percentage of control NOS activity. Data points represent mean±s.e.mean of five measurements from different animals. Vertical bars show s.e.mean. ^aP<0.05, ^bP<0.01 vs untreated control NOS activity. ^cP<0.05, ^dP<0.01 vs untreated UI NOS activity.