Vascular responses to 8-nitro-cyclic GMP in non-diabetic and diabetic mice

Abstract

Background and purpose: 8-Nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), formed nitric oxide (NO)-dependently, is a physiological second messenger, yet little is known about its role in the pathophysiology of vascular diseases. To study the pharmacological activity of 8-nitro-cGMP in diabetic mice, we compared its effects on vascular reactivity of aortas from non-diabetic and diabetic mice.

Experimental approach: Vascular tension recording was performed in thoracic aortic rings from wild-type (C57BL/6), non-diabetic db/+ and obese/diabetic db/db mice. Endothelial NO synthase (eNOS) uncoupling and superoxide were tested by Western blot and dihydroethidium fluorescence respectively.

Key results: 8-Nitro-cGMP, at concentrations up to 10 µM, enhanced phenylephrine-induced contractions in aortas from C57BL/6 and db/+ mice, but not from db/db mice. This enhancement was not observed with 8-bromo-cGMP. Pretreatment of aortas from C57BL/6 and db/+ mice with l-NAME (100 µM), superoxide dismutase (100 U·mL(-1) ) or tiron (1 mM), abolished 8-nitro-cGMP-induced enhancement of the phenylephrine contraction. In 8-nitro-cGMP (10 µM)-treated C57BL/6 aortas, eNOS dimer/monomer ratio was significantly decreased and vascular superoxide production increased, suggesting that 8-nitro-cGMP-induced superoxide production via eNOS uncoupling may mediate the enhancement of the phenylephrine contraction. At higher concentrations (>10 µM), 8-nitro-cGMP produced relaxation of the phenylephrine-contracted aortas from C57BL/6, db/+ and db/db mice. The 8-nitro-cGMP-induced relaxation in db/db mouse aortas was found to be resistant to a phosphodiesterase 5 inhibitor, zaprinast (1 µM).

Conclusions and implications: The vasodilator effect of 8-nitro-cGMP may contribute to amelioration of the vascular endothelial dysfunction in diabetic mice, representing a novel pharmacological approach to prevent the complications associated with diabetes.

Figure 1

Effects of l-NAME, SOD and tiron on 8-nitro-cGMP-induced enhancement of contraction to phenylephrine (Phe) in aortic rings from C57BL/6 mouse. Original tracings and summarized data of vascular responses to cumulatively administered 8-nitro-cGMP in the Phe-contracted aorta. Vertical lines in each trace indicate administration of 8-nitro-cGMP. 8-Nitro-cGMP concentration-dependently produced a biphasic effect: an initial enhancement of contraction to Phe, followed by a relaxation. l-NAME (0.1 mM, A, lower trace), SOD (100 U·mL⁻¹, B, upper trace) or tiron (1 mM, B, lower trace) was added 30 min before the addition of Phe (0.1 µM). l-NAME, SOD and tiron each abolished the enhancement of the contraction to Phe. Changes in vascular tension to 8-nitro-cGMP are expressed as % of the Phe-induced contraction. Each point represents the mean ± SEM (n = 4–6). *P < 0.05 versus control.

Figure 2

Disruption of eNOS dimer and superoxide production by 8-nitro-cGMP in aortas from C57BL/6 mouse. (A) Representative Western blots and densitometric analysis for eNOS dimer (280 KDa) and monomer (140 KDa) in C57BL/6 mouse aortic segments treated with vehicle, 8-nitro-cGMP or 8-bromo-cGMP. Each eNOS density was normalized to β-actin (45 KDa). (B) Representative fluorescent photomicrographs and quantitative analysis of DHE-labelled microscopic sections of C57BL/6 mouse aortic segments incubated with vehicle, 8-nitro-cGMP or 8-bromo-cGMP. Each value represents the mean ± SEM (n = 3–4). *P < 0.05 versus control and 8-bromo-cGMP.

Figure 3

Vascular reactivity in aortas from db/+ and db/db mice. (A) Phe (0.1 µM)-induced contraction of thoracic aortic rings from db/+ and db/db mice. Data are expressed as % of the tension induced by 50 mM KCl. (B) Cumulative concentration–relaxation curves to acetylcholine (ACh) of Phe-contracted aortas from db/+ and db/db mice (n = 6 for each group). Relaxations are expressed as a percentage reversal of the contraction induced by Phe (1 µM). Data are shown as mean ± SEM. *P < 0.02 versus db/+.

Figure 4

Effects of 8-nitro-cGMP on the contraction to Phe in the aortas from db/+ and db/db mice. Original tracings (A and B) and summarized data (C and D) of vascular responses to cumulatively administered 8-nitro-cGMP and 8-bromo-cGMP in the Phe-contracted aortas from db/+ and db/db mice. Vertical lines in each trace indicate administration of 8-nitro-cGMP (A) or 8-bromo-cGMP (B). Changes in vascular tension to 8-nitro-cGMP (C) and 8-bromo-cGMP (D) are expressed as % of the Phe (0.1 µM)-induced contraction. Each point represents the mean ± SEM (n = 5–9 for each group). *P < 0.05 versus db/+.

Figure 5

Effects of l-NAME, SOD and tiron on 8-nitro-cGMP-induced enhancement of the contraction to Phe in aortas from db/+ mice. l-NAME (0.1 mM, A), SOD (100 U·mL⁻¹, B) or tiron (1 mM, B) was added 30 min before the addition of Phe. Changes in vascular tension to 8-nitro-cGMP are expressed as % of the Phe-induced contraction in db/+ mouse aorta. Each point represents the mean ± SEM (n = 4–6). *P < 0.05 versus control.

Figure 6

Effects of zaprinast on 8-nitro-cGMP or 8-bromo-cGMP-induced relaxation in db/db aorta contracted by Phe. Cumulative concentration–relaxation curves to 8-nitro-cGMP (A) or 8-bromo-cGMP (B) of the Phe-contracted aortas from db/db mice (n = 4–6 for each group). Zaprinast (1 µM) was added 30 min before the addition of Phe. (C) Comparison of the relaxation responses to 8-nitro-cGMP (100 µM) and 8-bromo-cGMP (100 µM) induced in the presence and absence of zaprinast in db/db aortic rings contracted by Phe. Data are shown as mean ± SEM. *P < 0.05 versus control.

Figure 7

Hypothetical scheme for the mechanism that mediates the vascular tone induced by 8-nitro-cGMP. Stimulation of α₁-adrenoceptors by Phe increases intracellular Ca²⁺ concentrations ([Ca²⁺]_i), leading to contraction of vascular smooth muscle cells. In vascular endothelial cells, concomitantly, eNOS is activated by the increase in [Ca²⁺]_i induced by α₁-adrenoceptor stimulation to produce NO and induce vascular relaxation via PKG activation. In the non-diabetic mouse aortas, 8-nitro-cGMP applied extracellularly may induce uncoupling (black line) of the eNOS activated by the Phe-induced increase in [Ca²⁺]_i, and may produce superoxide (·O²⁻), resulting in enhancement of the Phe-induced contraction. In addition, not only PKG activation via 8-nitro-cGMP itself, but also 8-RS-cGMP, which is formed by protein S-guanylation and is resistant to PDE5, may induce a potentially strong PKG activation and then vasorelaxation. The rank order of potency for the effect of 8-nitro-cGMP at lower concentrations up to 10 µM is contraction > relaxation. However, the rank order reverses at higher concentrations of 8-nitro-cGMP. In the diabetic mouse aorta with eNOS uncoupling (red arrow), the relaxation via NO generation is depressed (red arrow) and superoxide is produced (red arrow), and thus the amplitude of the contraction induced by Phe is increased (red oval and seesaw-like balance). 8-Nitro-cGMP, applied in the diabetic aorta, does not induce eNOS uncoupling (broken blue line) and thus does not enhance Phe-contraction (blue oval), but induces only relaxation via PKG activation. The relaxant effect of 8-nitro-cGMP on the enhancement of vasoconstriction might be beneficial in compensating for excess oxidative stress (blue arrows), such as endothelial dysfunction of diabetic mice.