Therapeutic concentrations of raloxifene augment nitric oxide-dependent coronary artery dilatation in vitro

Abstract

Background and purpose: Raloxifene improves cardiovascular function. This study examines the hypothesis that therapeutic concentrations of raloxifene augment endothelium-dependent relaxation via up-regulation of eNOS expression and activity in porcine coronary arteries.

Experimental approach: Isometric tension was measured in rings from isolated arteries. Intracellular Ca(2+) concentrations ([Ca(2+)](i)) in arterial endothelial cells were detected by Ca(2+) fluorescence imaging. Phosphorylation of eNOS at Ser-1177 was assayed by Western blot analysis.

Key results: In arterial rings pre-contracted with 9,11-dideoxy-11alpha,9alpha-epoxy-methano-prostaglandin F(2alpha) (U46619), treatment with raloxifene (1-3 nM) augmented bradykinin- or substance P-induced relaxation and this effect was antagonized by ICI 182,780, an estrogen receptor antagonist. The enhanced relaxation was abolished in rings treated with inhibitors of nitric oxide/cyclic GMP-dependent dilation, N(G)-nitro-L-arginine methyl ester (L-NAME) plus 1H-[1,2,4]oxadizolo[4,3-a]quinoxalin-1-one (ODQ). In contrast, effects of raloxifene were unaffected after inhibition of endothelium-derived hyperpolarizing factors by charybdotoxin plus apamin. Raloxifene (3 nM) did not influence endothelium-independent relaxation to sodium nitroprusside. 17beta-Estradiol (3-10 nM) also enhanced bradykinin-induced relaxation, which was inhibited by ICI 182,780. Treatment with raloxifene (3 nM) did not affect bradykinin-stimulated rise in endothelial cell [Ca(2+)](i). Raloxifene, 17beta-estradiol, and bradykinin increased eNOS phosphorylation at Ser-1177 and ICI 182,780 prevented effects of raloxifene or 17beta-estradiol but not that of bradykinin. Raloxifene had neither additive nor antagonistic effects on 17beta-estradiol-induced eNOS phosphorylation.

Conclusions and implications: Raloxifene in therapeutically relevant concentrations augmented endothelial function in porcine coronary arteries in vitro through ICI 182,780-sensitive mechanisms that were associated with increased phosphorylation of eNOS but independent of changes in endothelial cell [Ca(2+)](i).

Figure 1

Two consecutive concentration-response curves (DRC) for bradykinin-induced relaxation in U46619-precontracted isolated porcine coronary arteries with endothelium in control (a), in the presence of 1 nM (b) or 3 nM (c) raloxifene (Rf). Results are mean±s.e.mean of 6–8 experiments. ^*P<0.05.

Figure 2

The effect of 3 nM raloxifene (Rf) on bradykinin-induced relaxation in rings with endothelium, in the absence (a) and presence of ICI 182780 (ICI, b). (c) Inhibition by ICI 182780 on the enhancing effect of raloxifene (3 nM) on substance P-induced relaxation. (d) Lack of effect of raloxifene on A23187-induced relaxation. Results are mean±s.e.mean of 6–8 experiments. ^*P<0.05 between Rf and other groups.

Figure 3

Effect of 3 nM raloxifene (Rf) on bradykinin-induced relaxation in rings with endothelium, in the absence and presence of treatment with N^G-nitro-L-arginine methyl ester(L-NAME) plus 1H-[1,2,4]oxadizolo[4,3-a]quinoxalin-1-one (ODQ) (a) or with charybdotoxin (CTX) plus apamin (b). (c) L-NAME/ODQ- or (d) CTX/apamin-insensitive component of bradykinin relaxation. Results are mean±s.e.mean of six experiments. ^*P<0.05 between control and Rf groups (two-way ANOVA).

Figure 4

(a) Two identical relaxation curves to bradykinin in rings with endothelium. (b) Enhancing relaxation to bradykinin in the presence of 3 or 10 nM 17β-estradiol (E₂). (c) Abolition by ICI 182780 of 17β-estradiol (10 nM)-induced enhanced relaxation to bradykinin. (d) Lack of effect of ICI 182780 alone on bradykinin-induced relaxation. Results are mean±s.e.mean of 5–7 experiments. ^*P<0.05 between control and estrogen groups (two-way ANOVA).

Figure 5

Calcium levels in en face preparations of endothelial cells of intact porcine coronary arteries measured by ratiometric fluorescence imaging. Images show increases in [Ca²⁺]_i in vitro induced by bradykinin (BK, 100 nM) in the absence (a) and presence (b) of 3 nM raloxifene (Rf). Increases in [Ca²⁺]_i are expressed as averaged area under curve in response to bradykinin with and without raloxifene treatment (c). Results are mean±s.e.mean of experiments from six different pigs.

Figure 6

Effects of raloxifene (3 nM), 17β-estradiol (3 nM), raloxifene (3 nM) plus 17β-estradiol (3 nM), and bradykinin (50 nM) on total eNOS protein expression levels (a) and eNOS phosphorylation at Ser-1177 (b) with and without treatment with 1 μM ICI 182780. Results are mean±s.e.mean of five experiments on arteries from different pigs. Statistical difference is indicated by ^*(P<0.001) between groups.