Role of inducible nitric oxide synthase in endothelium-independent relaxation to raloxifene in rat aorta

Abstract

Background and purpose: Raloxifene can induce both endothelium-dependent and -independent relaxation in different arteries. However, the underlying mechanisms by which raloxifene triggers endothelium-independent relaxation are still incompletely understood. The purpose of present study was to examine the roles of NOSs and Ca²⁺ channels in the relaxant response to raloxifene in the rat isolated, endothelium-denuded aorta.

Experimental approach: Changes in isometric tension, cGMP, nitrite, inducible NOS protein expression and distribution in response to raloxifene in endothelium-denuded aortic rings were studied by organ baths, radioimmunoassay, Griess reaction, western blot and immunohistochemistry respectively.

Key results: Raloxifene reduced the contraction to CaCl₂ in a Ca²⁺ -free, high K⁺ -containing solution in intact aortic rings. Raloxifene also acutely relaxed the aorta primarily through an endothelium-independent mechanism involving NO, mostly from inducible NOS (iNOS) in vascular smooth muscle layers. This effect of raloxifene involved the generation of cGMP and nitrite. Also, it was genomic in nature, as it was inhibited by a classical oestrogen receptor antagonist and inhibitors of RNA and protein synthesis. Raloxifene-induced stimulation of iNOS gene expression was partly mediated through activation of the NF-κB pathway. Raloxifene was more potent than 17β-estradiol or tamoxifen at relaxing endothelium-denuded aortic rings by stimulation of iNOS.

Conclusions and implications: Raloxifene-mediated vasorelaxation in rat aorta is independent of a functional endothelium and is mediated by oestrogen receptors and NF-κB. This effect is mainly mediated through an enhanced production of NO, cGMP and nitrite, via the induction of iNOS and inhibition of calcium influx through Ca²⁺ channels in rat aortic smooth muscle.

Figure 1

(A) The time course for the relaxant response induced by different concentrations of raloxifene (Rf; 0.1–3 μM) in the U46619‐contracted endothelium‐intact aortic rings. (B) The steady‐state maximum relaxation to raloxifene. Statistical differences are indicated by between vehicle control and raloxifene group (*P < 0.05). Results are mean ± SEM of six to seven rings from different rats. (C) The inhibitory effects of L‐NAME (100 μM) and ODQ (3 μM) on raloxifene (1 μM)‐induced relaxation in endothelium‐intact rat aortic rings. Treatment with indomethacin (Indo, 3 μM) was without effect. (D) Steady‐state maximum relaxation induced by raloxifene after various treatments. Rings were exposed for 30 min to each inhibitor before addition of U46619 to induce a sustained tone. Statistical differences are indicated by ^a between vehicle control and raloxifene group and ^b between raloxifene and other treatment groups (*P < 0.05). Results are mean ± SEM of 6–10 rings from different rats. (E) Raloxifene‐induced relaxant effect was similar in endothelium‐intact aortic rings contracted by U46619 and 60 mM K⁺. (F) Steady‐state maximum relaxation induced by raloxifene after various treatments. Results are mean ± SEM of five to six rings from different rats. (G) Concentration‐dependent inhibition by raloxifene (0.1–3 μM) on CaCl₂‐induced contractile responses in Ca²⁺‐free, 60 mM K⁺‐containing Krebs solution. (H) pEC₅₀ values for CaCl₂‐induced contraction in the absence and presence of raloxifene (0.1–1 μM). *P < 0.05 between control and all treatment curves for CaCl₂ responses. Results are mean ± SEM of five to six rings from different rats.

Figure 2

(A) Recordings showing the time course of raloxifene‐induced relaxation in endothelium‐denuded aortic rings in control and in the presence of 100 μM L‐NAME and 3 μM ODQ. (B) Inhibitory effect of L‐NAME on raloxifene (1 μM)‐induced aortic relaxation and L‐arginine (L‐Arg, 0.5–1 mM) antagonized the effect of L‐NAME. (C) The inhibitory effect of ODQ on raloxifene (1 μM)‐induced aortic relaxation and lack of effect of 1 mM L‐arginine. (D) The maximal relaxant effect of raloxifene following different pharmacological manipulations. All experiments were performed on endothelium‐denuded rings. Statistical differences are indicated by *(P < 0.05) between vehicle control and raloxifene group, ^a (P < 0.05) between raloxifene and other treatment groups and ^b (P < 0.05) between L‐NAME and treatment groups. Results are mean ± SEM of 6–11 rings from different rats.

Figure 3

(A) Recordings showing the time course of raloxifene‐induced relaxation in endothelium‐denuded aortic rings in control and in the presence of 100 μM AMT‐HCl or 100 μM aminoguanidine. Inhibitory effect of AMT‐HCl (B) or aminoguanidine (C) on raloxifene (1 μM)‐induced aortic relaxation. (D) The maximal relaxant effect of raloxifene in the absence and presence of iNOS inhibitors. (E) Treatment with NPLA (100 μM) was without effect on raloxifene‐induced relaxation. (F) The maximal relaxant effect of raloxifene in the absence and presence of NPLA. Statistical differences are indicated by * (P < 0.05) between vehicle control and raloxifene group, ^a (P < 0.05) between raloxifene and other treatment groups and ^b (P < 0.05) between AMT‐HCl/aminoguanidine and treatment groups. Results are mean ± SEM of 6–11 rings from different rats.

Figure 4

(A) Recordings showing the time course of raloxifene‐induced relaxation in endothelium‐denuded aortic rings in control and in the presence of 10 μM actinomycin D (ACT‐D) or 10 μM cycloheximide (CHX). (B) Inhibitory effect of actinomycin D or cycloheximide on raloxifene (1 μM)‐induced aortic relaxation. (C) The maximal relaxant effect of raloxifene in the absence and presence of actinomycin D/cycloheximide. Statistical differences are indicated by * (P < 0.05) between vehicle control and raloxifene group and ^a (P < 0.05) between raloxifene and other treatment groups. Results are mean ± SEM of 6–11 rings from different rats.

Figure 5

(A) Recordings showing time‐dependent relaxant responses to 1 μM raloxifene in endothelium‐denuded aortic rings in control and in the presence of 1 μM ICI 182780. (B) Inhibitory effect of ICI 182780 on raloxifene‐induced aortic relaxation. (C) The maximal relaxant effect of raloxifene in the absence and presence of ICI 182780. Results are mean ± SEM of six to seven rings from different rats. (D) Recordings showing the time course of raloxifene‐induced relaxation in endothelium‐denuded aortic rings in control and in the presence of 3 μM PDTC. (E) Inhibitory effects of PDTC on relaxant responses to raloxifene in endothelium‐denuded aortic rings. (F) The maximal relaxant effect of raloxifene in the absence and presence of PDTC. Results are mean ± SEM of six rings from different rats. Statistical differences are indicated by * (P < 0.05) between vehicle control and raloxifene group and ^a (P < 0.05) between raloxifene and other treatment groups.

Figure 6

(A) Raloxifene‐stimulated increases in cyclic GMP (cGMP) levels in endothelium‐denuded rat aortic rings and effects of various inhibitors (L‐NAME, ODQ, aminoguanidine, AMT, actinomycin D and cyclohexinide). The effect of LPS (10 μg·mL⁻¹) served as a positive control. Statistical differences (P < 0.05) are indicated by ^a between control and treatment groups and ^b between raloxifene (1 μM) and other treatment groups. Results are mean ± SEM of six rings from different rats. (B) Basal and raloxifene‐stimulated release of nitrite and the effects of pharmacological inhibitors on raloxifene‐induced increase in nitrite production. All experiments were performed on endothelium‐denuded rat aortic rings. The effect of LPS (10 μg·mL⁻¹) or SNP (100 nM) served as a positive control. Statistical differences (P < 0.05) are indicated by ^a between control and treatment groups and ^b between raloxifene (1 μM) and other treatment groups. Results are mean ± SEM of six to eight rings from different rats.

Figure 7

(A) Effects of inhibitors on the increase in iNOS protein expression induced by 1 μM raloxifene after pre‐contracting with 20 nM U46619 in endothelium‐denuded rings. Representative western blot analysis (top panel). Densitometry analysis of results of western blots (bottom panel). Statistical differences are indicated between control and treatment groups, and between raloxifene (1 μM) and other treatment groups (*P < 0.05). Data are expressed as the value of integrated density value (IDV) mg^‐1 protein. The results are means ± SEM of 6 to 12 experiments. The position of iNOS with 135 kDa was verified by molecular markers and LPS‐treated rat macrophage NR 8383 cell lysate. (B) Effects of inhibitors on the increase in iNOS protein expression induced by 1 μM raloxifene after pre‐contracting with 20 nM U46619 in endothelium‐denuded rings in vascular smooth muscle. Representative immunohistochemistry sections of endothelium‐denuded rings. Area stained with red colour (like raloxifene and LPS group) represents the location of iNOS induction. Original magnification: ×400. Scale bar: 100 μm. Figure is representative of at least three experiments performed on different experimental days.